Respiratory syncytial virus recombinant fusion proteins having prefusion conformations, methods for their preparation and uses - Patent Application 20070122999

Abstract

This application relates to a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a host cell transformed or transfected with the polynucleotide, nucleic acid construct, or expression vector, a stabilized trimer formed therefrom, an immunogenic composition containing any of the foregoing, and its use in preparing a vaccine for preventing and / or treating respiratory syncytial virus infection. The RSV-B recombinant F protein of this application contains at least one specific epitope of a pre-fusion F protein and can form a stabilized F protein trimer with a pre-fusion conformation, which is stable in expression, has a uniform morphology, and significantly improved yield. The formed F protein trimer has excellent immunogenicity and can stimulate the body to produce high levels of antibody titers, which is of great significance for the clinical treatment, prevention, and control of respiratory syncytial virus.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed on September 29, 2022, bearing application number 202211199603.X and entitled "Respiratory syncytial virus recombinant fusion protein with pre-fusion conformation, preparation method and use thereof," the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present application relates to the field of vaccines, and in particular to a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, stabilized trimers formed therewith, immunogenic compositions containing same, methods for their preparation and use. [Background technology]

[0003] Respiratory syncytial virus (RSV) is the most common pathogen causing acute respiratory infections in infants. Bronchiolitis caused by RSV infection is one of the leading causes of hospitalization in children under 2 years of age and is a significant factor contributing to increased infant mortality. According to WHO estimates, 64 million children worldwide are infected with RSV each year, of which 160,000 die from RSV infection. In 2020, the number of children under 5 years of age with severe RSV infection worldwide reached 34.6 million, with 3 million infected in China alone. Another high-risk group for RSV infection is the elderly. Several prospective studies have reported the incidence of RSV infection among elderly people living in housing complexes, finding that the annual incidence of RSV infection ranges from 2% to 10%. Large-scale epidemiological studies have shown that RSV causes hospitalization and mortality rates in the elderly comparable to those of influenza.

[0004] RSV has only a single serotype with two major antigenic subgroups, A and B. Strains of both subtypes often coexist and circulate, but only one subtype usually predominates during epidemics. In temperate regions, including China, RSV infections exhibit clear seasonality, occurring from late autumn to early winter, peaking from mid-December to early February, and beginning to decline in late spring. Safe, effective, and affordable preventive and therapeutic strategies against RSV infections are urgently needed worldwide.

[0005] RSV, a member of the Pneumovirus genus in the Paramyxoviridae family, is a polymorphic enveloped virus with a diameter of approximately 120–300 nm. It possesses a non-segmented, negative-sense, single-stranded RNA (15–16 kb) encoding 11 proteins. The viral envelope contains three proteins: the attachment glycoprotein (G), the fusion glycoprotein (F), and the small hydrophobic (SH) protein. The G protein functions in attachment to host cells, the F protein is involved in fusion and cell entry, and the SH protein is not required for either process. Two RSV surface glycoproteins are major neutralizing antigens. The G protein exhibits high sequence diversity and determines the antigenic subtypes (A and B) of the virus. The fusion protein (F) is highly conserved between the two groups of subtypes and is recognized by broadly cross-neutralizing antibodies. Due to its important role in RSV entry and its highly conserved protein sequence, the RSV F protein is the target of neutralizing antibodies and the primary antigen for vaccine development. The F protein is a type I transmembrane protein. It is initially synthesized as a 574-amino acid precursor protein, F0, which contains five to six post-translational N-linked glycosylation modifications. To become a functional fusion protein, F0 is processed by a cellular furin-like protease at two polybasic sites in the trans-Golgi apparatus to generate F1 (amino acids 137–574), F2 (amino acids 26–109), and Pep27 polypeptides, ultimately forming two disulfide-linked fragments, F1 and F2, which are 55 kD and 15 kD in size, respectively. The homologous F protein trimer contains two conformations: pre-fusion and post-fusion. The pre-fusion F protein is a metastable structure, and upon virus fusion with the cell, it is converted into a stable post-fusion F protein; this process can also occur spontaneously. Because the highly active neutralizing antibody epitopes against the F protein are mainly located on the pre-fusion F protein, how to maintain the F protein in the pre-fusion conformation is the key to developing an RSV vaccine, and to date, no RSV vaccine is available on the market. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the problems present in the prior art described above, the present application provides a respiratory syncytial virus subtype B (RSV-B) recombinant F protein (abbreviated herein as "RSV-B-Fm" or "Fm"), a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell transformed or transfected with the polynucleotide, nucleic acid construct, or expression vector, a stabilized trimer formed by the recombinant F protein, an immunogenic composition comprising any of the above, and its use in preparing a vaccine for preventing and / or treating respiratory syncytial virus infection. [Means for solving the problem]

[0007] Specifically, the present application provides the following technical solutions: In a first aspect, the present application provides a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, which is a recombinant F protein based on respiratory syncytial virus subtype B, also referred to herein as a RSV-B recombinant protein or a RSV-B recombinant F protein, wherein the RSV-B recombinant F protein comprises a RSV-B-F1 peptide fragment and a RSV-B-F2 peptide fragment, wherein the RSV-B-F1 peptide fragment and / or the RSV-B-F2 peptide fragment comprises at least one proline mutation relative to the corresponding peptide fragment of a wild-type RSV-B F protein.

[0008] In certain embodiments, the RSV-B-F1 peptide fragment corresponds to an amino acid fragment at positions 26-98, 26-108, or 26-97 of the amino acid sequence of the wild-type RSV-B F protein set forth in SEQ ID NO: 1, and the RSV-B-F2 peptide fragment corresponds to an amino acid fragment at positions 136-513, 145-513, 138-513, or 137-513 of the amino acid sequence of the wild-type RSV-B F protein set forth in SEQ ID NO: 1, and the RSV-B-F1 peptide fragment and / or the RSV-B-F2 peptide fragment comprises one or more proline mutations relative to the corresponding peptide fragment of the wild-type RSV-B F protein.

[0009] Preferably, the one or more proline mutations are selected from the group consisting of: a mutation selected from K65P, T67P, D73P, L138P, G139P, L141P, E161P, Q210P, I214P, S215P, N216P, Q279P, S377P; Preferably, the one or more mutations to prolines are For example, T67P in the RSV-B recombinant F protein Fm 1 shown in SEQ ID NO: 29, For example, L141P in the RSV-B recombinant F protein Fm2 shown in SEQ ID NO: 30, For example, Q279P in the RSV-B recombinant F protein Fm 3 shown in SEQ ID NO: 31, For example, S377P in the RSV-B recombinant F protein Fm4 shown in SEQ ID NO: 32, For example, T67P+L141P in the RSV-B recombinant F protein Fm 5 shown in SEQ ID NO: 33, For example, L141P+Q279P in the RSV-B recombinant F protein Fm 6 shown in SEQ ID NO: 34, For example, L141P+S377P in the RSV-B recombinant F protein Fm 7 shown in SEQ ID NO: 35; For example, T67P+Q279P in the RSV-B recombinant F protein Fm 8 shown in SEQ ID NO: 36; For example, T67P+S377P in the RSV-B recombinant F protein Fm 9 shown in SEQ ID NO: 37, For example, Q279P+S377P in the RSV-B recombinant F protein Fm 10 shown in SEQ ID NO: 38; For example, T67P+L141P+Q279P in the RSV-B recombinant F protein Fm 11 shown in SEQ ID NO: 39; For example, T67P+L141P+S377P in the RSV-B recombinant F protein Fm 12 shown in SEQ ID NO: 40; For example, L141P+Q279P+S377P in the RSV-B recombinant F protein Fm 13 shown in SEQ ID NO: 41; For example, T67P+Q279P+S377P in the RSV-B recombinant F protein Fm 14 shown in SEQ ID NO: 42; For example, T67P+L138P+G139P in the RSV-B recombinant F protein Fm 15 shown in SEQ ID NO: 43; For example, L138P+G139P+Q279P in the RSV-B recombinant F protein Fm 16 shown in SEQ ID NO: 44; For example, L138P+G139P+S377P in the RSV-B recombinant F protein Fm 17 shown in SEQ ID NO: 45; For example, T67P+L138P+G139P+Q279P in the RSV-B recombinant F protein Fm 18 shown in SEQ ID NO: 46; For example, T67P+L138P+G139P+S377P in the RSV-B recombinant F protein Fm 19 shown in SEQ ID NO: 47; For example, L138P + G139P + Q279P + S377P in the RSV-B recombinant F protein Fm 20 shown in SEQ ID NO: 48; For example, T67P+L141P+Q279P+S377P in the RSV-B recombinant F protein Fm 21 shown in SEQ ID NO: 49; For example, T67P+L138P+G139P+Q279P+S377P in the RSV-B recombinant F protein Fm 22 shown in SEQ ID NO: 50; For example, the mutation or combination of mutations selected from the following in the RSV-B recombinant F protein Fm 23 as set forth in SEQ ID NO: 51: T67P + L138P + G139P + L141P + Q279P + S377P; Optionally, in the above mutations or combinations of mutations, the T67P mutation can be replaced with a K65P mutation (e.g., RSV-B recombinant F protein Fm 24 as set forth in SEQ ID NO: 52) or a D73P mutation (e.g., RSV-B recombinant F protein Fm 25 as set forth in SEQ ID NO: 53); Optionally, in the above mutations or combinations of mutations, the L141P mutation may be replaced with a L138P mutation or a G139P mutation; Optionally, the above mutations or combinations of mutations further include one or more mutations selected from an N216P mutation, a Q210P mutation, an I214P mutation, an S215P mutation, and an E161P mutation.

[0010] For example, in some embodiments, for example, in RSV-B recombinant F protein Fm 26-34, whose amino acid sequences are set forth in any of SEQ ID NOs: 54-62, an N216P mutation is further introduced into the above mutations. In other embodiments, for example, in RSV-B recombinant F protein Fm 35, whose amino acid sequence is set forth in SEQ ID NO: 63, an Q210P mutation is further introduced into the above mutations. In other embodiments, for example, in RSV-B recombinant F protein Fm 36, whose amino acid sequence is set forth in SEQ ID NO: 64, an I214P mutation is further introduced into the above mutations. In other embodiments, for example, in RSV-B recombinant F protein Fm 37-38, whose amino acid sequences are set forth in SEQ ID NOs: 65-66, an S215P mutation is further introduced into the above mutations. In other embodiments, for example, in RSV-B recombinant F protein Fm 39-55, whose amino acid sequences are set forth in any of SEQ ID NOs: 67-83, an E161P mutation is further introduced into the above mutations.

[0011] In some embodiments, for example, in RSV-B recombinant F protein Fm 74-76 (eg, as set forth in SEQ ID NOs: 102-104), the RSV-B-F1 peptide fragment and the RSV-B-F2 peptide fragment are directly linked.

[0012] In some other embodiments, the RSV-B-F1 peptide fragment and the RSV-B-F2 peptide fragment are linked via a linker, Preferably, the linker is (i) Optionally, a (GS)m linker (where m = 1 to 5, preferably 1 to 3) containing one or more amino acid mutations, for example, as shown in SEQ ID NO: 9 (e.g., in RSV-B recombinant F protein Fm 69 to 71 (shown in SEQ ID NOs: 97 to 99)), or SEQ ID NO: 10 (e.g., in RSV-B recombinant F protein Fm 72 to 73 (shown in SEQ ID NOs: 100 to 101)), (ii) A (GGGGS)n linker (where n = 1 to 5, preferably 1 to 3), optionally containing one or more amino acid mutations, such as mutations to proline, addition of amino acids, etc. In certain embodiments, this type of linker is represented by the sequence of SEQ ID NO: 2 (e.g., in RSV-B recombinant F protein Fm 1 to 55 (e.g., as set forth in SEQ ID NOs: 29 to 83)), SEQ ID NO: 3 (e.g., in RSV-B recombinant F protein Fm 56 to 59 (e.g., as set forth in SEQ ID NOs: 84 to 87)), SEQ ID NO: 4 (e.g., in RSV-B recombinant F protein Fm 60 (e.g., as set forth in SEQ ID NO: 88)), SEQ ID NO: 5 (e.g., in RSV-B recombinant F protein Fm 61 (e.g., as set forth in SEQ ID NO: 89)), or SEQ ID NO: 6 (e.g., in RSV-B recombinant F protein Fm 62 (e.g., as set forth in SEQ ID NO: 90)); (iii) the linking sequence of F0 itself, between the wild-type F1 and F2 peptide fragments, optionally the sequence shown in SEQ ID NO: 7 (e.g., in the RSV-B recombinant F protein Fm 63-65 (e.g., SEQ ID NOs: 91-93)); and (iv) A sequence obtained by mutating the furin cleavage site in the linker (iii), optionally selected from the sequence shown in SEQ ID NO: 8 (e.g., in RSV-B recombinant F protein Fm 66 to 68 (e.g., SEQ ID NOs: 94 to 96)).

[0013] Preferably, the linker comprises or consists of an amino acid sequence shown in SEQ ID NOs: 2 to 10.

[0014] In a preferred specific embodiment, the RSV-B recombinant F protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 104, or amino acid sequences having similar or substantially similar immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence set forth in any of SEQ ID NOs: 29 to 104.

[0015] Furthermore, in some embodiments, the RSV-B recombinant F protein further comprises a trimerization 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 NOs: 24 to 27, Optionally, a His tag as shown in SEQ ID NO: 28 may be added to the C-terminus for subsequent protein isolation and purification.

[0016] Furthermore, in some embodiments, the RSV-B recombinant F protein further comprises 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 NOs: 11-23.

[0017] In a second aspect, the present application provides a polynucleotide encoding the RSV-B recombinant F protein according to the first aspect above.

[0018] In a particular embodiment, the polynucleotide is a humanized codon-optimized nucleotide sequence, which may be DNA or mRNA; In some embodiments, the polynucleotide is a DNA molecule, and preferably, the DNA molecule comprises or consists of a DNA sequence set forth in any one of SEQ ID NOs: 105-180.

[0019] In some other embodiments, the polynucleotide is an mRNA molecule, and preferably the mRNA molecule comprises or consists of an RNA sequence corresponding to the DNA sequence set forth in any of SEQ ID NOs: 105 to 180.

[0020] In a third aspect, the present application provides a nucleic acid construct comprising a polynucleotide according to the second aspect above, and optionally at least one expression control element operably linked to said polynucleotide.

[0021] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid construct according to the third aspect above.

[0022] In a fifth aspect, the present application relates to a cell line transformed or transfected with a polynucleotide according to the second aspect above, a nucleic acid construct according to the third aspect above, or an expression vector according to the fourth aspect above, Optionally, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell; Further, optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell; Further, optionally, the host cell is provided wherein said bacterial cell is an E. coli cell.

[0023] In a sixth aspect, the present application provides a respiratory syncytial virus subtype B (RSV-B) recombinant F protein trimer, which is a polymerized version of three RSV-B recombinant F proteins described in the first aspect above.

[0024] In a seventh aspect, the present application provides the use of a RSV-B recombinant F protein according to the first aspect above, a polynucleotide according to the second aspect above, a nucleic acid construct according to the third aspect above, an expression vector according to the fourth aspect above, a host cell according to the fifth aspect above, or a RSV-B recombinant F protein trimer according to the sixth aspect above, in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection.

[0025] In an eighth aspect, the present application provides a vaccine or immunogenic composition comprising a RSV-B recombinant F protein according to the first aspect above, a polynucleotide according to the second aspect above, a nucleic acid construct according to the third aspect above, an expression vector according to the fourth aspect above, a host cell according to the fifth aspect above, or a RSV-B recombinant F protein trimer according to the sixth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, vector and / or diluent.

[0026] In some preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, comprising the RSV-B recombinant F protein described in the first aspect above or the RSV-B recombinant F protein trimer described in the sixth aspect above, and an adjuvant; Optionally, the adjuvant is one or more selected from aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and AS-series-like adjuvant.

[0027] In some other preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus DNA vaccine; (1) a eukaryotic expression vector; (2) A DNA sequence encoding the RSV-B recombinant F protein described in the first aspect above, constructed in the eukaryotic expression vector, preferably a DNA sequence shown in any one of SEQ ID NOs: 105 to 180; Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS, and pcDNA series vectors.

[0028] In some other preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising: (I) an mRNA sequence encoding the RSV-B recombinant F protein described in the first aspect above, preferably an mRNA sequence corresponding to any one of the DNA sequences set forth in SEQ ID NOs: 105 to 180; (II) lipid nanoparticles.

[0029] In another preferred specific embodiment, the vaccine or immunogenic composition is a respiratory syncytial virus-viral vector vaccine, (1) a viral backbone vector; (2) A DNA sequence encoding the RSV-B recombinant F protein described in the first aspect above, constructed in the viral backbone vector, preferably a DNA sequence shown in any one of SEQ ID NOs: 105 to 180; Optionally, the viral backbone vector is one or more selected from an adenovirus vector, a poxvirus vector, an influenza virus vector, and an adeno-associated virus vector.

[0030] In a possible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation; Preferably, the nasal drops are selected from aerosols, sprays, and powder aerosols; Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film-coated tablets, pellets, sublingual tablets, and plasters; Preferably, the parenteral formulation is a transdermal formulation, an ointment, a plaster, a topical solution, an injection or a bolus.

[0031] In a ninth aspect, the present application provides a method for manufacturing a semiconductor device comprising: The present invention provides a method for preparing the RSV-B recombinant F protein according to the first aspect, comprising: adding a nucleotide sequence encoding a signal peptide to the 5' end of a codon-optimized nucleotide sequence encoding the RSV-B recombinant F protein according to the first aspect; adding nucleotide sequences encoding a trimer tag and a histidine tag and a stop codon to the 3' end of the nucleotide sequence; cloning and expression; screening for a correct recombinant; transfecting the recombinant into an expression cell for expression; collecting the cell culture supernatant; and isolating the RSV-B recombinant F protein therefrom.

[0032] In one possible implementation of the above method, the expression system cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell, and optionally, the bacterial cell is an E. coli cell. [Effects of the Invention]

[0033] The beneficial effects are as follows: When native RSV-B virus F protein antigen is recombinantly expressed in vitro, it is not possible to obtain an F antigen protein with a pre-fusion conformation due to the instability of the pre-fusion conformation. In the present application, one or more proline mutations are introduced into the F1 and / or F2 peptide fragment of the F protein of respiratory syncytial virus type B to form a stable RSV-B recombinant F protein with a pre-fusion conformation. Experimental results show that the formed RSV-B recombinant F protein contains at least one specific epitope of the pre-fusion F protein, and is stable in expression, has a uniform morphology, and significantly improved yield. Furthermore, the present RSV-B recombinant F protein has excellent immunogenicity and can stimulate the body to produce high levels of antibody titers (the antigen-specific antibody titers induced by booster immunization with the recombinant protein without the addition of adjuvant all reach above 10,000, while the antigen-specific antibody titers can be further increased by 10-100 times after the addition of AddaVax adjuvant), which is of great significance for the clinical treatment, prevention, and control of respiratory syncytial virus. [Brief explanation of the drawings]

[0034] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative illustrations are not intended to be limiting of the embodiments. As used herein, the term "exemplary" means "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments. [Figure 1] In Example 3, the expression status of RSV-B recombinant F protein in the raw supernatant was detected by ELISA using the D25 monoclonal antibody, where the abscissa is the experimental group (i.e., the RSV-B recombinant F protein to be detected) and the ordinate is the absorbance value at OD450nm. [Figure 2]In Example 3, the expression status of RSV-B recombinant F protein in the supernatant was detected by ELISA using palivizumab, where the abscissa is the experimental group (i.e., the RSV-B recombinant F protein to be detected), and the ordinate is the absorbance value at OD450nm. [Figure 3] In Example 3, the expression status of a portion of the RSV-B recombinant F protein in the diluted supernatant was detected by ELISA using the D25 monoclonal antibody, where the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the right figure shows the experimental group (i.e., the RSV-B recombinant F protein to be detected). [Figure 4] In Example 3, the expression status of a portion of the RSV-B recombinant F protein in the diluted supernatant detected by ELISA using palivizumab is shown, where the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the right figure shows the experimental group (i.e., the RSV-B recombinant F protein to be detected). [Figure 5] In Example 4, the supernatant from which the RSV-B recombinant F protein Fm41 was expressed (supernatant from which the RSV-B recombinant F protein Fm41 was expressed) was purified by molecular sieve chromatography, and the purified protein was subjected to analytical ultracentrifugation.The figure on the left shows the ultraviolet absorption chart of molecular sieve chromatography used to purify the supernatant from which the RSV-B recombinant F protein Fm41 was expressed, and the figure on the right shows the results of analytical ultracentrifugation of the purified RSV-B recombinant F protein Fm41. [Figure 6] 1 shows an ultraviolet absorption chart of the expression supernatant of RSV-B recombinant F protein Fm23 purified by molecular sieve chromatography in Example 4. [Figure 7] 1 is an ultraviolet absorption chart of Example 4, in which the expression supernatant of RSV-B recombinant F protein Fm54 is purified by molecular sieve chromatography. [Figure 8]In Example 6, the titer levels of specific antibody IgG in mouse serum after priming and boosting mice with the RSV-B recombinant F protein Fm41 described in Example 5 as an immunogen are shown, where the abscissa is the grouping described in Table 1 and the ordinate is the antibody titer, as detected by ELISA assay. [Figure 9] In Example 8, the expression of RSV-B-Fm 1 to 10 and WT recombinant F proteins in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, as well as the binding status of each recombinant F protein to each of the above antibodies. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 10] In Example 8, the expression of RSV-B-Fm 11-20 recombinant F proteins in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 11] In Example 8, the expression of RSV-B-Fm 21-30 recombinant F protein in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 12]In Example 8, the expression of RSV-B-Fm 31-40 recombinant F protein in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 13] In Example 8, the expression of RSV-B-Fm 41-50 recombinant F protein in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, and the ordinate is the absorbance value at OD450nm. The experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 14] In Example 8, the expression of RSV-B-Fm 51-60 recombinant F protein in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, and the ordinate is the absorbance value at OD450nm. The experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 15] In Example 8, the expression of RSV-B-Fm 61-70 recombinant F protein in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, and the ordinate is the absorbance value at OD450nm. The experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 16] In Example 8, the expression of RSV-B-Fm 71-76 recombinant F proteins in diluted supernatants was detected by ELISA using D25, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies, and the binding status of each recombinant F protein to each of the above antibodies was shown. Here, the abscissa is the Log10 value of the dilution ratio of the cell supernatant, the ordinate is the absorbance value at OD450nm, and the experimental group (i.e., the RSV-B recombinant F protein to be detected) is shown in the right figure. [Figure 17] The results of SDS-PAGE detection of RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 recombinant F proteins after separation and purification in Example 9 are shown. [Figure 18] 1 shows the results of Western blot detection of RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 recombinant F proteins after separation and purification in Example 9. [Figure 19] In Example 10, the binding status of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins to antibodies detected by ELISA using the D25 monoclonal antibody before and after 4 weeks of storage at 30°C is shown, where the abscissa is protein concentration and the ordinate is absorbance at OD450nm. [Figure 20] In Example 10, the antibody binding status of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins detected by ELISA using palivizumab before and after 4 weeks of storage at 30°C is shown, where the abscissa is protein concentration and the ordinate is absorbance at OD450nm. [Figure 21] In Example 12, the titer levels of specific binding antibody IgG in the serum of mice after priming (A) and boosting (B) with the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 54, 70, 75, and 76 recombinant F proteins described in Example 11, as detected by ELISA assay, are shown, where the abscissa is the vaccine group and the ordinate is the antibody titer. [Figure 22]In Example 13, the viral load in lung tissue of mice immunized with the vaccine described in Example 11 and challenged intranasally with respiratory syncytial virus is shown, as detected by plaque assay, where the abscissa is the vaccine group and the ordinate is the viral load. DETAILED DESCRIPTION OF THE INVENTION

[0035] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described below clearly and completely, but it is clear that the described embodiments are only a part of the embodiments of the present application, and are not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without any creative work fall within the scope of protection of the present application.

[0036] In addition, in order to better explain the present application, many specific details are described in the following specific embodiments. It should be understood that those skilled in the art can practice the present application without certain specific details. In some examples, in order to clarify the gist of the present application, materials, elements, methods, means, etc. that are well known to those skilled in the art are not described in detail.

[0037] Unless expressly stated otherwise, throughout the specification and claims, the terms "comprises" or "comprises" or variations thereof such as "comprising" are to be understood to include the stated elements or components and not to exclude other elements or components. Example 1: Construction of the expression plasmid for the present RSV-B recombinant F protein

[0038] In this example, 76 types of RSV-B recombinant F proteins of the present invention were designed and constructed, and designated RSV-B-Fm 1 to 76, respectively, and their amino acid sequences are shown in SEQ ID NOs: 29 to 104, respectively.

[0039] The nucleic acid sequences encoding the RSV-B recombinant F proteins RSV-B-Fm 1 to 76 of the present application were optimized according to the codon preferences of mammalian cells to obtain the optimized nucleic acid coding sequences shown in SEQ ID NOs: 105 to 180, respectively. An EcoRI restriction enzyme site sequence, a Kozak sequence, and a nucleic acid coding sequence for a signal peptide (shown in SEQ ID NOs: 11 to 23) were added to the 5' end of each of the nucleic acid coding sequences shown in SEQ ID NOs: 105 to 180, respectively. A nucleic acid coding sequence for a trimer tag (shown in SEQ ID NOs: 24 to 27), a nucleic acid coding sequence for a His tag (shown in SEQ ID NO: 28), a stop codon, and an XhoI restriction enzyme site sequence were then added to the 3' end. Gene synthesis was then outsourced to GenScript Biotech, Inc., and the resulting product was ligated into the pCAGGS vector via the EcoRI and XhoI restriction enzyme sites to obtain expression plasmids for expressing the recombinant proteins RSV-B-Fm 1 to 76.

[0040] The signal peptides and trimerization tags used in each RSV-B recombinant F protein are as follows:

[0041] Fm1 to Fm37 (whose amino acid sequences are shown in SEQ ID NOs: 29 to 65, respectively, and whose optimized nucleic acid coding sequences are shown in SEQ ID NOs: 105 to 141, respectively) and Fm39 to Fm76 (whose amino acid sequences are shown in SEQ ID NOs: 67 to 104, respectively, and whose optimized nucleic acid coding sequences are shown in SEQ ID NOs: 143 to 180, respectively) all use the signal peptide shown in SEQ ID NO: 14 and the trimer tag shown in SEQ ID NO: 24.

[0042] Fm38 (amino acid sequence shown in SEQ ID NO: 66 and optimized nucleic acid coding sequence shown in SEQ ID NO: 142) uses a signal peptide shown in SEQ ID NO: 12 and a trimer tag shown in SEQ ID NO: 25. Example 2: Expression and isolation and purification of detection antibodies

[0043] In this example, the heavy and light chain expression plasmids for the D25 monoclonal antibody, an RSVF protein detection antibody, and palivizumab were constructed according to the antibody heavy and light chain sequences and the method for constructing the expression plasmids disclosed in the literature (Z. Wei et al., Analytical Chemistry 79, 2797-2805 (2007); Q. Zhu et al., Science Translational Medicine 9, (2017)). Antibody expression

[0044] 14–16 h prior to transfection, densely packed 293T cells were split into plates (e.g., a 10 cm dish filled with 100% 293T cells was passaged at a 1:3 ratio). Transfection was performed 14–16 h later, when cell density reached 70% or higher. Antibody heavy and light chain plasmids were co-transfected into 293T cells at a 2:3 ratio. 4–6 h after transfection, cells were washed twice with PBS and cultured in serum-free DMEM medium. Cell supernatants were collected on days 3 and 7 post-transfection, respectively, and cellular debris was removed by centrifugation. The two antibody supernatants were combined and used for subsequent antibody protein purification. Antibody purification

[0045] A Protein A (5 ml) HP affinity column (GE) was connected to an AKTA Purifier / Explorer / FPLC / START (GE). The instrument was operated as follows: First, the 20% ethanol in the column was washed out with water. Then, the column was equilibrated with 20 mM Na3PO4, pH 7.0 buffer. Once the instrument's conductivity stabilized, the antibody supernatant was loaded using a 10 ml loop to bind to Protein A. The flow rate was set to 2 ml / min. After UV stabilization, approximately 0.8 ml of 1 M Tris, pH 9.0 buffer was added to the next collection tube (collection volume approximately 3.2 ml). The program was changed to elute the column-bound antibody with 100% 0.1 M Gly, pH 3.0. The eluate was collected, and the antibody buffer was replaced with PBS by concentration and liquid exchange. The resulting antibody solution was either used directly or aliquoted and stored in a -80°C refrigerator for later use. Example 3: Expression and conformational identification of RSV-B-Fm recombinant protein

[0046] In this example, the expression plasmid for a portion of the RSV-B-Fm recombinant protein (Fm12-76) constructed in Example 1 was transfected into HEK293T cells to express the RSV-B-Fm recombinant protein. The conformation of the expressed recombinant protein was determined by ELISA using the detection antibody D25 monoclonal antibody prepared in Example 2 and palivizumab.

[0047] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS, and the cell density was adjusted to 70% or higher before transfection. The RSV-B-Fm recombinant protein Fm12-76 expression plasmids constructed in Example 1 were transfected into HEK293T cells. Four to six hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the cells were cultured for three days. The cell culture supernatant was collected, and antigen protein expression was detected by ELISA using D25 monoclonal antibody and palivizumab, respectively. The specific detection method is as follows:

[0048] (1) The purified detection antibodies (D25 monoclonal antibody and palivizumab) obtained in Example 2 were each diluted to 1 μg / ml with ELISA coating solution (Solebol, C1050), and 100 μl was added per well to a 96-well ELISA plate (Coring, 3590). The plate was then left at 4°C for 12 hours.

[0049] (2) The coating solution was discarded, and PBS was added and washed once. 200 μl of 5% skim milk prepared with PBS was added per well of a 96-well plate as a blocking solution, and the plate was left at room temperature for 1 hour. After blocking was complete, the plate was washed once with PBS.

[0050] (3) During the blocking period in step (2), the cell culture supernatant was diluted with blocking solution, starting from 5-fold and then diluted in a 3-fold gradient. Then, 100 μl of the undiluted culture supernatant of each RSV-B recombinant F protein to be detected or the culture supernatant of each dilution of a portion of the RSV-B recombinant F protein (Fm22, 23, 41, 51, 52, 53, 54, 57) was added to each well of the ELISA plate. For the negative control, blocking solution was added, and the plate was incubated at 37°C for 2 hours, followed by washing four times with PBST.

[0051] (4) HRP-labeled anti-His antibody (purchased from MBL) was added and incubated at 37°C for 1.5 hours, followed by washing 5-6 times with PBST. Then, TMB color development solution was added to develop color. After the appropriate reaction time, 2M hydrochloric acid was added to stop the reaction, and the OD450 reading was detected using a microplate reader.

[0052] The expression results of each RSV-B recombinant F protein in the supernatant are shown in Figures 1 and 2. As can be seen from the results shown in Figures 1 and 2, the expression of all detected RSV-B-Fm recombinant proteins was significantly improved compared to NC and wild-type RSV-B F proteins, and all detected RSV-B-Fm recombinant proteins could bind to palivizumab (which recognizes F proteins with both pre- and post-fusion conformations) and the D25 monoclonal antibody (which recognizes only F proteins with a pre-fusion conformation). This indicates that the RSV-B-Fm recombinant proteins of the present application are in a pre-fusion conformation.

[0053] The expression results of RSV-B recombinant F proteins Fm22, 23, 41, 51, 52, 53, 54, and 57 in the gradient diluted supernatants are shown in Figures 3 and 4. The results shown in Figures 3 and 4 show that when the RSV-B-Fm22, 23, 41, 51, 52, 53, 54, and 57 cell-expressed supernatants were diluted 1000-fold, both D25 monoclonal antibody and palivizumab could still detect the proteins, indicating that RSV-B-Fm22, 23, 41, 51, 52, 53, 54, and 57 had high expression levels. Example 4: Expression, purification and molecular weight determination of RSV-B-Fm recombinant protein

[0054] In this example, the RSV-B-Fm41, 23, and 54 recombinant protein expression plasmids constructed in Example 1 were transfected into HEK293T cells to express the RSV-B-Fm41, 23, and 54 recombinant proteins, which were then purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The target proteins were collected and subjected to analytical ultracentrifugation to determine the molecular weight of the expressed recombinant proteins.

[0055] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS, and the cell density was adjusted to 70% or higher before transfection. The RSV-B-Fm41, 23, and 54 recombinant protein expression plasmids constructed in Example 1 were transfected into HEK293T cells. Four to six hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the cells were cultured for three days. The supernatant was then collected, and the DMEM medium was further replenished. The cells were cultured for four days, and the supernatant was again collected. The two harvests of cell culture supernatant were mixed and centrifuged at 5000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the target protein was bound to a HisTrap Excel column (5 mL, GE Healthcare). Nonspecifically bound proteins were 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 with an elution buffer containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 400 mM imidazole. The fractions containing the target protein were collected and concentrated, and then subjected to molecular sieve chromatography (Superdex 200 Increase 10 / 300GL or Superdex 200 Hiload 16 / 60, GE Healthcare) to obtain the purified RSV-B-Fm recombinant protein antigen. At the same time, the target protein peak was collected and subjected to analytical ultracentrifugation to determine the molecular weight of the expressed recombinant protein.

[0056] The UV absorption chart of the molecular sieve chromatography of the recombinant protein RSV-B-Fm41 is shown in the left panel of Figure 5. It was found that the elution peak of the recombinant protein RSV-B-Fm41 contained a small amount of a multimeric impurity peak in addition to the target protein peak. The target protein peak was collected and subjected to analytical ultracentrifugation, and the results are shown in the right panel of Figure 5. As can be seen from the right panel of Figure 5, the measured molecular weight of RSV-B-Fm41 was 170 kDa, which is consistent with the theoretical molecular weight of the trimer, indicating that the expressed RSV-B-Fm41 recombinant protein is in the form of a trimer.

[0057] The ultraviolet absorption charts of the molecular sieve chromatography of RSV-B-Fm23 and 54 are shown in Figures 6 and 7, respectively. Figures 6 and 7 show that the elution peak of RSV-B-Fm23 contains not only the target protein peak but also a small amount of a multimeric impurity peak, whereas the elution peak of RSV-B-Fm54 is almost a single peak and basically does not contain any impurity peaks. Example 5: RSV-B-Fm recombinant protein immunization mouse experiment

[0058] In this example, mice were immunized with the recombinant protein RSV-B-Fm41 obtained in Example 4. The experimental mice were BALB / c mice aged 4 to 6 weeks and weighing an average of 15 to 20 g.

[0059] Specifically, the BALB / c mice were immunized with the recombinant RSV-B-Fm41 protein obtained in Example 4, with or without the addition of an adjuvant, and the adjuvant was AddaVax, an MF59-like adjuvant. Also, the mice were immunized with the same dose of saline as a negative control.

[0060] The immunization group status, immunogen used in each group, immunogen dose used in each group, and adjuvant status are shown in Table 1, with blanks indicating "none." The RSV-B-Fm41 recombinant protein was diluted to the required concentration with saline, and the adjuvanted groups were further emulsified with adjuvant separately, with each group consisting of 6 mice.

[0061] [Table 1]

[0062] Mice in each group were intramuscularly injected with the recombinant protein vaccine or saline at a volume of 100 μl per immunization on days 0 and 14. Blood was collected from the tail vein on days 13 and 28, respectively. The mouse blood was allowed to stand and then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then inactivated (incubated at 56°C for 30 minutes), aliquoted, and stored in a refrigerator at -80°C. Example 6: Detection of vaccine-induced specific antibody titers by ELISA assay

[0063] In this example, the titers of specific IgG antibodies in the sera of mice immunized with the RSV-B-Fm41 recombinant protein of Example 5 were detected by ELISA assay.

[0064] Specifically, the following procedure is adopted: (1) The RSV-B-Fm41 recombinant protein prepared in Example 4 was diluted to 3 μg / ml with ELISA coating solution (Solabo, C1050), and 100 μl was added per well of a 96-well ELISA plate (Coring, 3590), followed by incubation at 4°C for 12 hours.

[0065] (2) The coating solution was discarded, and PBS was added and washed once. 100 μl of 5% skim milk prepared with PBS was added per well of a 96-well plate as a blocking solution, and the plate was left at room temperature for 1 hour for blocking. After blocking was complete, the plate was washed once with PBS.

[0066] (3) During the blocking period in step (2), mouse serum samples were diluted with blocking solution, starting from 10-fold and then diluted 2-fold. Then, 100 μl of the immune serum dilution solution was added per well to the ELISA plate, and blocking solution was added to the negative control. The plate was incubated at 37°C for 2 hours, followed by washing four times with PBST.

[0067] (4) HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 in blocking solution was added and incubated at 37°C for 1 hour, followed by washing 5-6 times with PBST. TMB color development solution was added, and after the appropriate reaction time, the reaction was stopped by adding 2M hydrochloric acid, and the OD450 reading was detected using a microplate reader.

[0068] The antibody titer was defined as the highest serum dilution at which the response value exceeded 2.1 times the negative control value. If the response value at the lowest dilution (detection limit) was still below 2.1 times the background value, the titer of this sample was defined as half the lowest dilution, i.e., 1:5.

[0069] The results are shown in Figure 8. The results in Figure 8 demonstrate that the RSV-B-Fm41 recombinant protein has excellent immunogenicity. When booster immunizations were performed with the recombinant protein without the addition of adjuvant, the induced antigen-specific antibody titers were all above 10,000. However, after the addition of AddaVax adjuvant, the antigen-specific antibody titers could be further increased by 10 to 100 times. Example 7: Expression and isolation and purification of detection antibodies

[0070] The heavy and light chain expression plasmids for the RSVF protein detection antibodies AM14, MPE8, 101F and hRSV90 monoclonal antibodies were constructed according to the antibody heavy and light chain sequences and the construction methods of their expression plasmids 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 Microbiol2, 16272 (2017); Fabian Sesterhenn., et al. Science 368, eaay5051 (2020); Mousa, J., et al. Nat Microbiol2, 16271 (2017)).

[0071] For specific methods of antibody expression, isolation and purification, see Example 2. Finally, the obtained antibody solution can be used directly or stored in a refrigerator at -80°C in small portions for later use. Example 8: Expression and conformational identification of RSV-B-Fm recombinant protein

[0072] In this example, all of the RSV-B-Fm recombinant protein (Fm1-76) expression plasmids constructed in Example 1 were transfected into HEK293T cells to express the RSV-B-Fm recombinant proteins, which were then detected by ELISA using the detection antibodies prepared in Examples 2 and 7, thereby determining the expression level and conformation of the expressed recombinant proteins. For specific methods, see Example 3.

[0073] The expression results of each RSV-B recombinant F protein in the supernatant are shown in Figures 9 to 16. Figures 9 to 16 show that, compared to the wild-type RSV-B F protein, the expression of RSV-B-Fm2, 6, 11 to 20, 21 to 24, 29, 31, 33 to 37, 39, 40, 41 to 45, 51 to 53, 55 to 60, 61 to 65, 69, 70, and 71 to 76 recombinant F proteins was significantly improved. Furthermore, these recombinant F proteins were able to bind to the corresponding monoclonal antibody for shared epitope II (i.e., palivizumab) and the corresponding monoclonal antibody for shared epitope IV (i.e., 101F monoclonal antibody) before and after fusion. It was also found that the RSV-B-Fm2, 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 application are in the prefusion conformation. Example 9: Expression, purification and SDS-PAGE identification of RSV-B-Fm recombinant protein

[0074] In this example, the RSV-B-Fm36, 37, 38, 52, 53, 70, 75, and 76 recombinant protein expression plasmids constructed in Example 1 were transfected into HEK293T cells to express the RSV-B-Fm36, 37, 38, 52, 53, 70, 75, and 76 recombinant proteins, which were then purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). Specific operating steps are described in Example 4.

[0075] The target protein peak was collected by gel filtration chromatography and subjected to SDS-PAGE analysis. The results are shown in Figure 17. Figure 17 shows that the RSV-B-Fm36, 37, 38, 52, 53, 70, 75, and 76 recombinant proteins all showed a single clear protein band, all of which were highly pure and had molecular weights between 55 KDa and 70 KDa, consistent with the theoretical molecular weight.

[0076] Furthermore, because all RSV-B-Fm recombinant F proteins are recombinant proteins with a histidine tag, in this example, an anti-histidine tag antibody conjugated with horseradish peroxidase was also used for Western blot identification, and the results are shown in Figure 18. Figure 18 shows that expression of the histidine tag was detected in each RSV-B-Fm recombinant F protein collection, indicating that the expression of these RSV-B-Fm recombinant F proteins was consistent with expectations and that all were correctly expressed. Example 10: Stability detection of RSV-B-Fm recombinant protein

[0077] The RSV-B-Fm36, 37, 52, 53, 75, and 76 recombinant proteins obtained in Examples 4 and 9 were sterile filtered and stored at 30°C. After 4 weeks, the antigen-antibody binding was measured by ELISA using D25 monoclonal antibody (targeting epitope φ) and palivizumab (targeting epitope II), respectively, to determine the stability of the proteins during long-term storage. The specific detection method is as follows:

[0078] (1) The purified antibodies (palivizumab and D25 monoclonal antibodies) obtained in Examples 2 and 8 were each diluted to 1 μg / ml with ELISA coating solution (Solebol, C1050), and 100 μl of each was added to each well of a 96-well ELISA plate (Corning, 3590). The plate was then left at 4°C for 12 hours.

[0079] (2) The coating solution was discarded, and PBS was added and washed once. 200 μl of 5% skim milk prepared with PBS was added per well of a 96-well plate as a blocking solution, and the plate was left at room temperature for 1 hour. After blocking was complete, the plate was washed once with PBS.

[0080] (3) During the blocking period in step (2), the RSV-B-Fm recombinant protein was diluted in blocking solution, starting from 300 ng / ml and diluted three-fold. Then, 100 μl of the diluted RSV-B-Fm recombinant protein was added to each well of the ELISA plate. For the negative control, blocking solution was added. The plate was incubated at 37°C for 2 hours and then washed four times with PBST.

[0081] (4) HRP-labeled anti-His antibody (purchased from MBL) was added and incubated at 37°C for 1.5 hours, followed by washing 5-6 times with PBST. Next, TMB color development solution was added to develop color. After the appropriate reaction time, 2M hydrochloric acid was added to stop the reaction, and the OD450 reading was detected using a microplate reader.

[0082] The ELISA detection results using the D25 monoclonal antibody and palivizumab are shown in Figures 19 and 20, respectively. Figures 19 and 20 show that after storing the RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant proteins at 30°C for 4 weeks, there was no significant change in the binding of each protein to the D25 antibody and palivizumab, compared to the initial proteins at week 0, indicating that these RSV-B-Fm recombinant proteins have good stability. Example 11: Mouse immunization experiment with RSV-B-Fm recombinant protein

[0083] Mice were immunized with the RSV-B-Fm23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant proteins obtained in Examples 4 and 10. The experimental mice were BALB / c mice aged 6 to 8 weeks and weighing an average of 15 to 20 g.

[0084] Specifically, mice were immunized with the above-mentioned RSV-B-Fm recombinant F protein at an immunization dose of 12 μg each. The adjuvant was a composite adjuvant of aluminum hydroxide adjuvant (purchased from Croda) and CpG (purchased from Invivogen). The vaccine was prepared as follows: Each RSV-B-Fm recombinant protein was diluted to the required concentration with saline, and the aluminum hydroxide adjuvant was first mixed with the antigen protein, then with the CpG adjuvant. The placebo group received saline. Each group consisted of four 6- to 8-week-old BALB / c mice (average weight 15-20 g).

[0085] Mice in each group were intramuscularly injected with the recombinant protein vaccine or saline at a volume of 100 μl per immunization on days 0 and 14. Blood was collected from the tail vein on days 13 and 28, respectively. The mouse blood was allowed to stand and then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then inactivated (incubated at 56°C for 30 minutes) and then aliquoted and stored in a refrigerator at -80°C. Example 12: Detection of vaccine-induced antigen-specific antibody titers by ELISA assay

[0086] In this example, the titers (Log antibody titers) of specific IgG antibodies in the serum of mice immunized with the RSV-B-Fm23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant protein vaccines of Example 11 were detected by ELISA assay. For specific methods and definitions of antibody titers, see Example 6.

[0087] The results of measuring specific IgG antibody titers in mouse serum after the first and second immunizations are shown in Figures 21A and 21B, respectively. Figures 21A and 21B show that the antigen-specific antibody titers induced by all RSV-B-Fm recombinant F protein vaccines reached 10,000 or more after the first immunization, and further increased to 1,000,000 or more after the second immunization. This indicates that RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant F proteins all have excellent immunogenicity. Example 13: Evaluation of vaccine challenge protective efficacy

[0088] For mice immunized with each vaccine in Example 11, 10 4 Mice were challenged with the Long strain of respiratory syncytial virus via nasal instillation at a challenge volume of 50 μl per mouse. Five days after challenge, the mice were euthanized and the lungs were removed. After weighing and recording the lungs of four mice per group, DMEM medium was added, homogenized using a tissue homogenizer, and centrifuged at 5000 g / min for 10 minutes to obtain lung tissue supernatant. The amount of respiratory syncytial virus in the lung tissue supernatant samples was detected by plaque assay, and the number of viral copies per gram of lung tissue was finally calculated.

[0089] The specific method for the plaque assay is as follows: 1 x 10 BHK cells were plated in a 12-well plate one day prior to the assay. 5100 cells were seeded onto the cells, and a 10-fold dilution of the original tissue solution was added to the starting well: 50 μl of lung tissue supernatant was added to 450 μl of DMEM medium and mixed well. Six 10-fold dilutions were then made. The cells to be infected were washed twice with PBS, and a sample of the diluted lung tissue supernatant was taken and infected with 400 μl per well. The cells were incubated at 37°C for 2 hours. After that, the cells were washed with PBS, and 1 ml of an equal volume mixture of 2% sodium carboxymethylcellulose and 2X DMEM was added per well. The cells were then incubated at 37°C for 4 days. The cells were washed once with PBS, fixed with methanol for 10 minutes at room temperature, washed once with PBS, blocked with 5% skim milk in PBST at 37°C for 1 hour, incubated with palivizumab diluted with 5% skim milk at 37°C for 1 hour, washed three times with PBST (PBS containing 0.05% Tween 20), and then incubated with horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (purchased from Beyotime Biotechnology) at 37°C for 1 hour. The cells were washed three times with PBST (PBS containing 0.05% Tween 20) and incubated with AEC substrate (purchased from BD Biosciences). Brown spots were considered positive spots, and the PFU values ​​for each sample were counted.

[0090] The results are shown in Figure 22, which shows that after the placebo-immunized group was challenged with the vaccine, high titers of respiratory syncytial virus were detected in the lung tissue, while no virus was detected in any of the vaccine-immunized groups. This indicates that the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant protein vaccines were all able to protect the body from respiratory syncytial virus infection after immunization and exerted excellent protective effects.

[0091] It should be noted that the above embodiments are only used to explain the technical solutions of the present application, and are not intended to limit the same. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art will understand that the technical solutions described in the above embodiments can be modified or some technical features can be replaced with equivalents, but these modifications or replacements will not deviate from the essence of the corresponding technical solutions and the spirit and scope of the technical solutions of the present application. [Industrial Applicability]

[0092] The recombinant RSV-B F protein of the present invention contains at least one specific epitope of the pre-fusion F protein and can form a stabilized F protein trimer with a pre-fusion conformation, which is stable in expression, has a uniform morphology, and significantly improves yield. The formed F protein trimer has excellent immunogenicity and can stimulate the body to produce high levels of antibodies, which is of great significance for the clinical treatment, prevention, and control of respiratory syncytial virus.

Claims

1. 1. A respiratory syncytial virus subtype B (RSV-B) recombinant F protein comprising: The RSV-B recombinant F protein is RSV-B-F １ Peptide fragments and RSV-B-F ２ RSV-BF peptide fragments, １ Peptide fragments and / or RSV-BF ２ A recombinant respiratory syncytial virus subtype B (RSV-B) F protein, wherein the peptide fragment contains at least one proline mutation relative to the corresponding peptide fragment of wild-type RSV-B F protein.

2. The RSV-B-F １ The peptide fragment corresponds to an amino acid fragment located at positions 26 to 98, 26 to 108, or 26 to 97 of the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO: 1, and ２ The peptide fragment corresponds to an amino acid fragment located at positions 136 to 513, 145 to 513, 138 to 513, or 137 to 513 of the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO: 1, and １ Peptide fragments and / or RSV-BF ２ 2. The RSV-B recombinant F protein of claim 1, wherein the peptide fragment contains one or more proline mutations relative to the corresponding peptide fragment of the wild-type RSV-B F protein.

3. The one or more proline mutations are selected from the group consisting of the following mutations in the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO: 1: a mutation selected from K65P, T67P, D73P, L138P, G139P, L141P, E161P, Q210P, I214P, S215P, N216P, Q279P, S377P; Preferably, the one or more mutations to prolines are T67P, L141P, Q279P, S377P, T67P+L141P, L141P+Q279P, L141P+S377P, T67P+Q279P, T67P+S377P, Q279P+S377P, T67P+L141P+Q279P, T67P+L141P+S377P, L141P+Q279P+S377P, T67P+Q279P+S377P, T67P+L138P+G139P, L138P+G139P+Q279P, L138P+G139P+S377P, T67P+L138P+G139P+Q279P, T67P+L138P+G139P+S377P, L138P+G139P+Q279P+S377P, T67P+L141P+Q279P+S377P, T67P+L138P+G139P+Q279P+S377P, T67P+L138P+G139P+L141P+Q279P+S377P mutations or combinations of mutations; Optionally, in the above mutations or combinations of mutations, the T67P mutation may be replaced with a K65P mutation or a D73P mutation; Optionally, in the above mutations or combinations of mutations, the L141P mutation may be replaced with an L138P mutation or a G139P mutation; 3. The RSV-B recombinant F protein of claim 2, wherein optionally, the mutation or combination of mutations further includes one or more mutations selected from the group consisting of an N216P mutation, a Q210P mutation, an I214P mutation, an S215P mutation, and an E161P mutation.

4. The RSV-B-F １ Peptide fragments and RSV-BF ２ The peptide fragments are linked directly or via a linker, Preferably, the linker is (i) a (GS)m linker optionally comprising one or more amino acid mutations, wherein m=1 to 5, preferably 1 to 3; (ii) a (GGGGS)n linker optionally comprising one or more amino acid mutations, wherein n=1 to 5, preferably 1 to 3; (iii) Wild type F １ and F ２ The linking sequence of F0 itself between the peptide fragments, and (iv) a sequence obtained by mutating the furin cleavage site in the linker (iii); More preferably, the linker comprises or consists of an amino acid sequence selected from SEQ ID NOs: 2 to 10. The RSV-B recombinant F protein according to any one of claims 1 to 3.

5. The RSV-B recombinant F protein according to any one of claims 1 to 4, characterized in that the recombinant F protein comprises or consists of an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 29 to 104, or amino acid sequences having similar or substantially similar immunogenicity to the amino acid sequences set forth in any of SEQ ID NOs: 29 to 104, obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences set forth in any of SEQ ID NOs: 29 to 104.

6. The recombinant F protein further comprises a trimerization tag; The RSV-B recombinant F protein according to any one of claims 1 to 5, characterized in that the trimerization tag is preferably located at the C-terminus and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 27.

7. The recombinant F protein further comprises a signal peptide; The RSV-B recombinant F protein according to any one of claims 1 to 6, characterized in that the signal peptide is preferably located at the N-terminus and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 23.

8. A polynucleotide encoding the RSV-B recombinant F protein of any one of claims 1 to 7.

9. the polynucleotide is a DNA molecule or an mRNA molecule; Preferably, the DNA molecule comprises or consists of a DNA sequence set forth in any of SEQ ID NOs: 105 to 180; 9. The polynucleotide of claim 8, wherein the mRNA molecule preferably comprises or consists of an RNA sequence corresponding to a DNA sequence set forth in any of SEQ ID NOs: 105 to 180.

10. 10. A nucleic acid construct comprising the polynucleotide of claim 8 or 9, and optionally at least one expression control element operably linked to said polynucleotide.

11. An expression vector comprising the nucleic acid construct of claim 10.

12. A host cell comprising: Transformed or transfected with the polynucleotide of claim 8 or 9, the nucleic acid construct of claim 10, or the expression vector of claim 11, Optionally, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell; Further, optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell; Further, optionally, the host cell is an E. coli cell.

13. A respiratory syncytial virus type B (RSV-B) recombinant F protein trimer, which is a polymerized version of three RSV-B recombinant F proteins according to any one of claims 1 to 7.

14. Use of the RSV-B recombinant F protein of any one of claims 1 to 7, the polynucleotide of claim 8 or 9, the nucleic acid construct of claim 10, the expression vector of claim 11, the host cell of claim 12, or the RSV-B recombinant F protein trimer of claim 13 in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection.

15. A vaccine or immunogenic composition comprising the RSV-B recombinant F protein of any one of claims 1 to 7, the polynucleotide of claim 8 or 9, the nucleic acid construct of claim 10, the expression vector of claim 11, the host cell of claim 12, or the RSV-B recombinant F protein trimer of claim 13, and a physiologically acceptable vehicle, adjuvant, excipient, vector and / or diluent.

16. A respiratory syncytial virus recombinant protein vaccine, comprising the RSV-B recombinant F protein according to any one of claims 1 to 7 or the RSV-B recombinant F protein trimer according to claim 13, and an adjuvant; 16. The vaccine or immunogenic composition of claim 15, optionally wherein the adjuvant is one or more selected from an aluminum adjuvant, an MF59 adjuvant, an MF59-like adjuvant, and an AS-series-like adjuvant.

17. A respiratory syncytial virus DNA vaccine, the DNA vaccine comprising: (i) a eukaryotic expression vector; (ii) a DNA sequence encoding the RSV-B recombinant F protein according to any one of claims 1 to 7, preferably a DNA sequence set forth in any one of SEQ ID NOs: 105 to 180, constructed in the eukaryotic expression vector; 16. The vaccine or immunogenic composition of claim 15, wherein the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS, and pcDNA series vectors.

18. A respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising: (I) an mRNA sequence encoding the RSV-B recombinant F protein according to any one of claims 1 to 7, preferably an mRNA sequence corresponding to any one of the DNA sequences set forth in SEQ ID NOs: 105 to 180; (II) a lipid nanoparticle.

19. a respiratory syncytial virus vector vaccine, (1) a viral backbone vector; (2) A DNA sequence encoding the RSV-B recombinant F protein according to any one of claims 1 to 7, preferably a DNA sequence shown in any one of SEQ ID NOs: 105 to 180, constructed in the viral backbone vector; 16. The vaccine or immunogenic composition of claim 15, optionally wherein the viral backbone vector is one or more selected from an adenovirus vector, a poxvirus vector, an influenza virus vector, an adeno-associated virus vector.

20. the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation; Preferably, the nasal drops are selected from aerosols, sprays, and powder aerosols; Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film-coated tablets, pellets, sublingual tablets, and plasters; 20. The vaccine or immunogenic composition according to any one of claims 15 to 19, wherein the parenteral formulation is preferably a transdermal formulation, an ointment, a plaster, a liquid for external use, an injection or a bolus formulation.

21. A nucleotide sequence encoding a signal peptide is added to the 5' end of the codon-optimized nucleotide sequence encoding the RSV-B recombinant F protein according to any one of claims 1 to 5, and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon are added to the 3' end of the nucleotide sequence, followed by cloning and expression. After screening for a correct recombinant, the recombinant is transfected into an expression cell for expression, and the cell culture supernatant is collected and the RSV-B recombinant F protein is isolated therefrom; Optionally, the expression system cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell; Further, optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell; 6. The method for preparing an RSV-B recombinant F protein according to any one of claims 1 to 5, further comprising optionally, said bacterial cells being Escherichia coli cells.

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