A respiratory syncytial virus recombinant fusion protein in pre-fusion conformation, its preparation method and uses

Abstract

The present invention relates to a respiratory syncytial virus subgroup 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 transformed or transfected with the above-mentioned polynucleotide, nucleic acid construct or expression vector, a stabilized trimer formed therefrom, an immunogenic composition comprising any one of the foregoing, and its use in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection. The RSV-B recombinant F protein of the present invention comprises at least one specific epitope of the prefusion F protein, can form a stable F protein trimer in the prefusion conformation, and has stable expression, uniform form and greatly improved yield; the formed F protein trimer has good immunogenicity, can stimulate the body to produce a high level of antibody titer, and has great significance for the clinical treatment and prevention and control of respiratory syncytial virus.

Description

[0001] Cross-reference

[0002] This application claims the priority of a Chinese patent application filed on September 29, 2022, with the application number 202211199603.X and the invention title "A recombinant fusion protein of respiratory syncytial virus with a pre-fusion conformation, its preparation method and uses", the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention relates to the field of vaccine technology, and specifically relates to a recombinant F protein of respiratory syncytial virus subtype B (RSV-B), a stabilized trimer formed therefrom, an immunogenic composition containing the same, and its preparation method and uses. Background art

[0004] Respiratory syncytial virus (RSV) is the most common pathogen causing acute respiratory infections in infants. Bronchiolitis caused by RSV infection is one of the main reasons for hospitalization of children under 2 years old and an important factor leading to an increase in infant mortality. According to WHO estimates, 64 million children are infected with RSV globally every year, and 160,000 children die from RSV infection. In 2020, the number of children under 5 years old with severe RSV infection globally reached 34.6 million, and the number of infected children in China alone reached 3 million. Another high-risk group for RSV is the elderly. Several prospective studies have counted the incidence of RSV in community-dwelling elderly people, and the results revealed that the annual incidence of RSV infection ranges between 2% and 10%. Large-scale epidemiological studies have revealed that the hospitalization and mortality rates caused by the RSV virus in the elderly population are comparable to those of influenza.

[0005] RSV has only a single serotype and has two main antigenic subgroups A and B. Strains of both subtypes usually co-circulate, but usually only one subtype dominates during the epidemic period. In temperate regions including China, RSV infection shows obvious seasonality, occurring from the end of autumn to the beginning of winter, peaking between mid-December and early February, and starting to decline at the end of spring. There is an urgent need worldwide for safe, effective and inexpensive preventive and therapeutic methods for RSV virus infection.

[0006] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the Paramyxoviridae family. It is a pleomorphic enveloped virus with a diameter of approximately 120–300 nm, having a non-segmented negative-sense single-stranded RNA (15–16 kb), which encodes 11 proteins. The viral envelope contains three proteins, namely the attachment glycoprotein (G), the fusion glycoprotein (F), and the small hydrophobic protein (SH). The G protein plays a role in host cell attachment, the F protein is responsible for fusion and cell entry, and the SH protein is not required in either of these two processes. The two surface glycoproteins of RSV are the main neutralizing antigens: the G protein has a 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 a wide range of cross-neutralizing antibodies. Due to its key role in RSV invasion and the high conservation of the protein sequence, the RSV F protein is the target of neutralizing antibodies and the main antigen for vaccine development. The F protein belongs to type I transmembrane proteins. The F protein first synthesizes a precursor protein F0 of 574 amino acids, and there are 5-6 post-translational modified N-linked glycosylation modifications on the F0 protein. To become a functional fusion protein, F0 is processed at two polybasic sites by cellular furin-like proteases in the trans-Golgi apparatus, generating F1 (amino acids 137–574), F2 (amino acids 26–109), and Pep27 polypeptide, and finally forming two fragments of F1 and F2 connected by disulfide bonds, with sizes of 55 kD and 15 kD respectively. The homotrimeric F protein contains two conformations, pre-fusion and post-fusion. The pre-fusion F protein is a metastable structure. When the virus fuses with the cell, the pre-fusion F converts to the stable post-fusion F protein, and this process can also occur spontaneously. Since the epitopes of highly active neutralizing antibodies against the F protein are mainly on the pre-fusion F protein, how to keep the F protein in the pre-fusion conformation is the key to RSV vaccine development. To date, there is still no marketed RSV vaccine. Summary of the Invention

[0007] To overcome the problems existing in the above-mentioned prior art, the present invention provides a recombinant F protein of respiratory syncytial virus subtype B (RSV-B) (hereinafter simply referred to as "RSV-B-Fm" or "Fm"), 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 above polynucleotide, nucleic acid construct or expression vector, a stabilized 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 preventing and / or treating respiratory syncytial virus infection.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a respiratory syncytial virus subgroup B (RSV-B) recombinant F protein, which is a recombinant F protein based on respiratory syncytial virus subgroup B, also referred to herein as RSV-B recombinant protein or RSV-B recombinant F protein, and the RSV-B recombinant F protein comprises RSV-B-F 1 peptide segment and RSV-B-F 2 peptide segment, wherein the RSV-B-F 1 peptide segment and / or RSV-B-F 2 peptide segment contains at least one mutation to proline relative to the corresponding peptide segment of the wild-type RSV-B F protein.

[0010] In a specific embodiment, the RSV-B-F 1 peptide segment corresponds to the amino acid fragment at positions 26-98, 26-108 or 26-97 in the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO:1, and the RSV-B-F 2 peptide segment corresponds to the amino acid fragment at positions 136-513, 145-513, 138-513 or 137-513 in the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO:1, and the RSV-B-F 1 peptide segment and / or RSV-B-F 2 peptide segment contains one or more mutations to proline relative to the corresponding peptide segment of the wild-type RSV-B F protein.

[0011] Preferably, the one or more mutations to proline are mutations selected from the following in the amino acid sequence of the wild-type RSV-B F protein shown in SEQ ID NO:1:

[0012] K65P, T67P, D73P, L138P, G139P, L141P, E161P, Q210P, I214P, S215P, N216P, Q279P, S377P;

[0013] Preferably, the one or more mutations to proline are selected from the following mutations or combinations of mutations:

[0014] T67P; for example, in the RSV-B recombinant F protein Fm 1 shown in SEQ ID NO:29;

[0015] L141P; for example, in the RSV-B recombinant F protein Fm 2 shown in SEQ ID NO:30;

[0016] Q279P; for example, in the RSV-B recombinant F protein Fm 3 shown in SEQ ID NO:31;

[0017] S377P; for example, in the RSV-B recombinant F protein Fm 4 as shown in SEQ ID NO:32;

[0018] T67P + L141P; for example, in the RSV-B recombinant F protein Fm 5 as shown in SEQ ID NO:33;

[0019] L141P + Q279P; for example, in the RSV-B recombinant F protein Fm 6 as shown in SEQ ID NO:34;

[0020] L141P + S377P; for example, in the RSV-B recombinant F protein Fm 7 as shown in SEQ ID NO:35;

[0021] T67P + Q279P; for example, in the RSV-B recombinant F protein Fm 8 as shown in SEQ ID NO:36;

[0022] T67P + S377P; for example, in the RSV-B recombinant F protein Fm 9 as shown in SEQ ID NO:37;

[0023] Q279P + S377P; for example, in the RSV-B recombinant F protein Fm 10 as shown in SEQ ID NO:38;

[0024] T67P + L141P + Q279P; for example, in the RSV-B recombinant F protein Fm 11 as shown in SEQ ID NO:39;

[0025] T67P + L141P + S377P; for example, in the RSV-B recombinant F protein Fm 12 as shown in SEQ ID NO:40;

[0026] L141P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm 13 as shown in SEQ ID NO:41;

[0027] T67P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm 14 as shown in SEQ ID NO:42;

[0028] T67P + L138P + G139P; for example, in the RSV-B recombinant F protein Fm 15 as shown in SEQ ID NO:43;

[0029] L138P + G139P + Q279P; for example, in the RSV-B recombinant F protein Fm 16 as shown in SEQ ID NO:44;

[0030] L138P + G139P + S377P; for example, in the RSV-B recombinant F protein Fm 17 as shown in SEQ ID NO:45;

[0031] T67P + L138P + G139P + Q279P; for example, in the RSV-B recombinant F protein Fm18 as shown in SEQ ID NO:46;

[0032] T67P + L138P + G139P + S377P; for example, in the RSV-B recombinant F protein Fm19 as shown in SEQ ID NO:47;

[0033] L138P + G139P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm20 as shown in SEQ ID NO:48;

[0034] T67P + L141P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm21 as shown in SEQ ID NO:49;

[0035] T67P + L138P + G139P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm 22 as shown in SEQ ID NO:50;

[0036] T67P + L138P + G139P + L141P + Q279P + S377P; for example, in the RSV-B recombinant F protein Fm 23 as shown in SEQ ID NO:51;

[0037] Optionally, the T67P mutation in the above mutations or mutation combinations can be replaced by the K65P mutation (for example, the RSV-B recombinant F protein Fm 24 as shown in SEQ ID NO:52) or the D73P mutation (for example, the RSV-B recombinant F protein Fm25 as shown in SEQ ID NO:53);

[0038] Optionally, the L141P mutation in the above mutations or mutation combinations can be replaced by the L138P mutation or the G139P mutation;

[0039] Optionally, the above mutations or mutation combinations further include one or more mutations selected from the following: N216P mutation, Q210P mutation, I214P mutation, S215P mutation, and E161P mutation.

[0040] For example, in some embodiments, the N216P mutation is further introduced into the above mutations, for example, in the RSV-B recombinant F protein Fm 26-34 with an amino acid sequence as shown in any one of SEQ ID NOs: 54-62; in other embodiments, the Q210P mutation is further introduced into the above mutations, for example, in the RSV-B recombinant F protein Fm35 with an amino acid sequence as shown in SEQ ID NO: 63; in other embodiments, the I214P mutation is further introduced into the above mutations, for example, in the RSV-B recombinant F protein Fm 36 with an amino acid sequence as shown in SEQ ID NO: 64; in other embodiments, the S215P mutation is further introduced into the above mutations, for example, in the RSV-B recombinant F protein Fm 37-38 with an amino acid sequence as shown in SEQ ID NOs: 65-66; in other embodiments, the E161P mutation is further introduced into the above mutations, for example, in the RSV-B recombinant F protein Fm 39-55 with an amino acid sequence as shown in any one of SEQ ID NOs: 67-83.

[0041] In some embodiments, the RSV-B-F 1 peptide segment and RSV-B-F 2 peptide segment are directly connected, for example, in the RSV-B recombinant F protein Fm 74-76 (as shown in SEQ ID NOs: 102-104).

[0042] In other embodiments, the RSV-B-F 1 peptide segment and RSV-B-F 2 peptide segment are connected by a linker bridge;

[0043] Preferably, the linker bridge is selected from:

[0044] (i) The (GS)m linker bridge, where 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 the RSV-B recombinant F protein Fm 69-71 (as shown in SEQ ID NOs: 97-99), or as shown in SEQ ID NO: 10, for example, in the RSV-B recombinant F protein Fm 72-73 (as shown in SEQ ID NOs: 100-101));

[0045] (ii) A (GGGGS)n linker, where n = 1 - 5, preferably 1 - 3; optionally, it contains one or more amino acid mutations, for example, mutations to proline, addition of amino acids, etc.; in a specific embodiment, a linker of this type is shown in the following sequences: SEQ ID NO:2 (for example, in the RSV-B recombinant F protein Fm 1 - 55 (shown as SEQ ID NO:29 - 83)), SEQ ID NO:3 (for example, in the RSV-B recombinant F protein Fm 56 - 59 (shown as SEQ ID NO:84 - 87)), SEQ ID NO:4 (for example, in the RSV-B recombinant F protein Fm 60 (shown as SEQ ID NO:88)), SEQ ID NO:5 (for example, in the RSV-B recombinant F protein Fm 61 (shown as SEQ ID NO:89)) or SEQ ID NO:6 (for example, in the RSV-B recombinant F protein Fm 62 (shown as SEQ ID NO:90));

[0046] (iii) Wild-type F 1 Between F 2 Peptide segments, F 0 Its own linking sequence, optionally, is the sequence shown as SEQ ID NO:7 (for example, in the RSV-B recombinant F protein Fm 63 - 65 (shown as SEQ ID NO:91 - 93));

[0047] (iv) A sequence obtained by mutating the furin cleavage site on linker (iii), optionally, is the sequence shown as SEQ ID NO:8 (for example, in the RSV-B recombinant F protein Fm 66 - 68 (shown as SEQ ID NO:94 - 96)).

[0048] Preferably, the linker comprises an amino acid sequence selected from, or consisting of: the sequences shown as SEQ ID NO:2 - 10.

[0049] In a preferred specific embodiment, the RSV-B recombinant F protein comprises an amino acid sequence selected from the group consisting of, or 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 one of SEQ ID NO:29 - 104 and obtained by substitution, deletion or addition of one or several amino acids.

[0050] In addition, in some embodiments, the RSV-B recombinant F protein further comprises a trimer tag;

[0051] Preferably, the trimeric tag is located at the C-terminus and has an amino acid sequence selected from the group consisting of SEQ ID NO: 24-27;

[0052] Optionally, a His tag as shown in SEQ ID NO: 28 can also be added to the C-terminus to facilitate the subsequent separation and purification of the protein.

[0053] In addition, in some embodiments, the RSV-B recombinant F protein further comprises a signal peptide;

[0054] 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.

[0055] In a second aspect, the present invention provides a polynucleotide encoding the RSV-B recombinant F protein as described in the first aspect above.

[0056] In a specific embodiment, the polynucleotide is a nucleotide sequence optimized for human codons and can be DNA or mRNA;

[0057] In some embodiments, the polynucleotide is a DNA molecule. Preferably, the DNA molecule comprises a DNA sequence as shown in any one of SEQ ID NO: 105-180, or consists of the same.

[0058] In some other embodiments, the polynucleotide is an mRNA molecule. Preferably, the mRNA molecule comprises an RNA sequence corresponding to the DNA sequence as shown in any one of SEQ ID NO: 105-180, or consists of the same.

[0059] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and optionally, at least one expression regulatory element operably linked to the polynucleotide.

[0060] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0061] In a fifth aspect, the present invention provides a host cell 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;

[0062] Optionally, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell;

[0063] Further optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell;

[0064] Further optionally, the bacterial cell is an Escherichia coli cell.

[0065] In a sixth aspect, the present invention provides a respiratory syncytial virus subgroup B (RSV-B) recombinant F protein trimer, which is formed by polymerization of three RSV-B recombinant F proteins as described in the first aspect above.

[0066] 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 preventing and / or treating respiratory syncytial virus infection.

[0067] In an eighth aspect, the present invention provides a vaccine or immunogenic composition, which comprises 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 a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0068] In some preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, which comprises 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;

[0069] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant and AS series-like adjuvants.

[0070] In some other preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus DNA vaccine, which comprises:

[0071] (1) a eukaryotic expression vector; and

[0072] (2) a DNA sequence encoding the RSV-B recombinant F protein as described in the first aspect above, which is preferably inserted into the eukaryotic expression vector and is one of the DNA sequences shown in SEQ ID NO: 105-180;

[0073] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0074] In some other preferred specific embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus mRNA vaccine, and the mRNA vaccine comprises:

[0075] (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

[0076] (II) a lipid nanoparticle.

[0077] In another preferred specific embodiment, the vaccine or immunogenic composition is a respiratory syncytial virus - viral vector vaccine, which comprises:

[0078] (1) a viral backbone vector; and

[0079] (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 the DNA sequence shown in any one of SEQ ID NO: 105-180;

[0080] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

[0081] In a feasible implementation manner, the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;

[0082] Preferably, the nasal spray is selected from aerosols, sprays and powder aerosols;

[0083] Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coatings, pellets, sublingual tablets and pastes;

[0084] Preferably, the parenteral preparation is a transdermal agent, ointment, plaster, external liquid agent, injectable or pushable preparation.

[0085] In a ninth aspect, the present invention provides a method for preparing the RSV-B recombinant F protein as described in the first aspect above, characterized in that the preparation method comprises:

[0086] At the 5' end of the codon-optimized nucleotide sequence encoding the RSV-B recombinant F protein as described in the first aspect above, add a nucleotide sequence encoding a signal peptide, and at the 3' end, add a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon, perform cloning and expression, screen for correct recombinants, and then transfect the expression system cells for expression. Collect the cell culture supernatant and isolate the RSV-B recombinant F protein therefrom.

[0087] In a 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.

[0088] Beneficial effects

[0089] When the native RSV-B virus F protein antigen is recombinantly expressed in vitro, due to the instability of the pre-fusion conformation, it is impossible to obtain the F antigen protein in the pre-fusion conformation. In the present invention, by introducing one or more mutations towards proline in the F 1 and / or F 2 peptide segments of the respiratory syncytial virus B type F protein, the stable, pre-fusion conformation RSV-B recombinant F protein of the present 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 moreover, its expression is stable, the form is uniform, and the yield is greatly improved; in addition, the RSV-B recombinant F protein of the present invention has good immunogenicity and can stimulate the body to produce a high level of antibody titer (after booster immunization with the recombinant protein without adjuvant, the induced antigen-specific antibody titers all reach more than 10,000, and after adding AddaVax adjuvant, the antigen-specific antibody titers can be further increased by 10 - 100 times), which has great significance for the clinical treatment and prevention and control of respiratory syncytial virus. Brief Description of the Drawings

[0090] One or more embodiments are illustrated by way of example in the pictures in the corresponding drawings, and these exemplary illustrations do not constitute a limitation on the embodiments. The special word "exemplary" here means "serving as an example, embodiment or illustrative". Any embodiment illustrated as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0091] Figure 1 Show in Example 3, using the D25 monoclonal antibody, detect the expression of RSV-B recombinant F protein in the supernatant stock solution by ELISA method, where the abscissa is the experimental group (i.e., the RSV-B recombinant F protein detected), and the ordinate is the absorbance value at OD450 nm.

[0092] Figure 2 In Example 3, palivizumab was used to detect the expression of RSV-B recombinant F protein in the undiluted supernatant by ELISA. Here, the abscissa represents the experimental group (i.e., the RSV-B recombinant F protein being detected), and the ordinate represents the absorbance value at OD450nm.

[0093] Figure 3 In Example 3, D25 monoclonal antibody was used to detect the expression of some RSV-B recombinant F protein in the diluted supernatant by ELISA. Here, the abscissa represents the Log10 value of the dilution factor of the cell supernatant, the ordinate represents the absorbance value at OD450 nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0094] Figure 4 In Example 3, palivizumab was used to detect the expression of some RSV-B recombinant F protein in the diluted supernatant by ELISA. Here, the abscissa represents the Log10 value of the dilution factor of the cell supernatant, the ordinate represents the absorbance value at OD450nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0095] Figure 5 In Example 4, the figure shows the results of purification of the expression supernatant of RSV-B recombinant F protein Fm41 by size exclusion chromatography and analytical ultracentrifugation of the purified protein. Here, the left figure is the ultraviolet absorption graph of the expression supernatant of RSV-B recombinant F protein Fm41 during purification by size exclusion chromatography, and the right figure is the result of analytical ultracentrifugation of the purified RSV-B recombinant F protein Fm41.

[0096] Figure 6 In Example 4, the ultraviolet absorption graph of the expression supernatant of RSV-B recombinant F protein Fm23 during purification by size exclusion chromatography is shown.

[0097] Figure 7 In Example 4, the ultraviolet absorption graph of the expression supernatant of RSV-B recombinant F protein Fm54 during purification by size exclusion chromatography is shown.

[0098] Figure 8 In Example 6, the ELISA experiment was used to detect the titer level of specific antibody IgG in the mouse serum after the primary immunization and booster immunization of mice using RSV-B recombinant F protein Fm 41 as an immunogen as described in Example 5. Here, the abscissa represents the grouping in Table 1, and the ordinate represents the antibody titer.

[0099] Figure 9In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 1-10 and WT recombinant F proteins in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. The abscissa is the Log10 value of the dilution factor of the cell supernatant, and the ordinate is the absorbance value at OD450 nm. The right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0100] Figure 10 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 11-20 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. The abscissa is the Log10 value of the dilution factor of the cell supernatant, and the ordinate is the absorbance value at OD450 nm. The right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0101] Figure 11 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 21-30 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. The abscissa is the Log10 value of the dilution factor of the cell supernatant, and the ordinate is the absorbance value at OD450 nm. The right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0102] Figure 12 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 31-40 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. The abscissa is the Log10 value of the dilution factor of the cell supernatant, and the ordinate is the absorbance value at OD450 nm. The right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0103] Figure 13In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 41-50 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. Among them, the abscissa is the Log10 value of the dilution factor of the cell supernatant, the ordinate is the absorbance value at OD450 nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0104] Figure 14 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 51-60 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. Among them, the abscissa is the Log10 value of the dilution factor of the cell supernatant, the ordinate is the absorbance value at OD450 nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0105] Figure 15 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 61-70 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. Among them, the abscissa is the Log10 value of the dilution factor of the cell supernatant, the ordinate is the absorbance value at OD450 nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0106] Figure 16 In Example 8, D25, AM14, palizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 71-76 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above-mentioned antibodies by ELISA. Among them, the abscissa is the Log10 value of the dilution factor of the cell supernatant, the ordinate is the absorbance value at OD450 nm, and the right figure legend shows the experimental group (i.e., the RSV-B recombinant F protein being detected).

[0107] Figure 17 In Example 9, the SDS-PAGE detection results of the purified RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 recombinant F proteins are shown.

[0108] Figure 18Figure 9 shows the Western Blot detection results of the RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 recombinant F proteins after separation and purification in Example 9.

[0109] Figure 19 Figure 10 shows the binding of the RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins to antibodies before and after storage at 30°C for 4 weeks using the D25 monoclonal antibody by ELISA. The abscissa represents the protein concentration, and the ordinate represents the absorbance value at OD450 nm.

[0110] Figure 20 Figure 11 shows the binding of the RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins to antibodies before and after storage at 30°C for 4 weeks using the palizumab antibody by ELISA. The abscissa represents the protein concentration, and the ordinate represents the absorbance value at OD450 nm.

[0111] Figure 21 Figure 12 shows the titers of specific binding antibody IgG in the sera of mice after primary immunization (A) and booster immunization (B) with the RSV-B-Fm23, 36, 37, 38, 41, 52, 53, 54, 70, 75, and 76 recombinant F proteins as immunogens in Example 11 detected by ELISA. The abscissa represents the vaccine groups, and the ordinate represents the antibody titers.

[0112] Figure 22 Figure 13 shows the viral loads in the lung tissues of mice after intranasal challenge with respiratory syncytial virus after vaccination with the vaccines described in Example 11 detected by plaque assay. The abscissa represents the vaccine groups, and the ordinate represents the viral loads. Detailed implementation manners

[0113] 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 some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0114] In addition, for a better illustration of the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can be implemented without some specific details. In some embodiments, details of raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0115] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or variations thereof such as "comprises" or "comprising" shall be understood to include the stated element or component, without excluding other elements or other components.

[0116] Example 1: Construction of expression plasmid of RSV-B recombinant F protein of the present invention

[0117] In this example, 76 RSV-B recombinant F proteins of the present invention were designed and constructed, which were respectively named RSV-B-Fm 1-76, and their amino acid sequences are respectively shown in SEQ ID NO: 29-104.

[0118] According to the codon preference of mammalian cells, the nucleic acid sequences encoding the above RSV-B recombinant F proteins of the present invention - RSV-B-Fm 1-76 were optimized to obtain optimized nucleic acid coding sequences, which are respectively shown in SEQ ID NO: 105-180; the EcoRI restriction site sequence, Kozak sequence, and nucleic acid coding sequence of the signal peptide (shown in SEQ ID NO: 11-23) were added to the 5' end of the nucleic acid coding sequences shown in SEQ ID NO: 105-180 respectively, and the nucleic acid coding sequence of the trimer tag (shown in SEQ ID NO: 24-27), the nucleic acid coding sequence of the His tag (shown in SEQ ID NO: 28), the stop codon, and the XhoI restriction site sequence were added to the 3' end. Then, GeneScript Biotech Corporation was entrusted for gene synthesis, and through the two restriction sites of EcoRI and XhoI, they were ligated to the pCAGGS vector to obtain the expression plasmids for expressing the recombinant proteins RSV-B-Fm 1-76.

[0119] The situations of the signal peptides and trimer tags used for each RSV-B recombinant F protein are as follows:

[0120] Fm 1-37 (amino acid sequences are respectively shown in SEQ ID NO: 29-65, and the optimized nucleic acid coding sequences are respectively shown in SEQ ID NO: 105-141) and Fm 39-76 (amino acid sequences are respectively shown in SEQ ID NO: 67-104, and the optimized nucleic acid coding sequences are respectively 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;

[0121] Fm 38 (the amino acid sequence is shown in SEQ ID NO: 66, and the optimized nucleic acid coding sequence is 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.

[0122] Example 2: Detection of antibody expression and isolation and purification

[0123] In this example, according to the methods for constructing the heavy and light chain sequences of antibodies and their 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)), the heavy and light chain expression plasmids of the detection antibody - D25 monoclonal antibody and palivizumab against RSV F protein were constructed respectively.

[0124] Antibody expression

[0125] 14 - 16 h before transfection, plate the cells with a relatively high density of 293T cells (for example, a 10 cm culture dish fully covered with 100% 293T cells is passaged at a ratio of 1:3). After 14 - 16 h, transfection can be carried out when the cell density reaches more than 70%. During transfection, co - transfect the heavy and light chain plasmids of the antibody into 293T cells at a ratio of 2:3. After 4 - 6 hours of transfection, wash the cells twice with PBS and then change to serum - free DMEM medium for continued culture; collect the cell supernatant on the 3rd and 7th days after transfection respectively, centrifuge to remove cell debris, and mix the antibody supernatants obtained twice for subsequent antibody protein purification.

[0126] Antibody purification

[0127] Connect a Protein A (5 ml) HP affinity column (GE) to AKTA Purifier / Explorer / FPLC / START (GE). The operation process on the instrument is as follows: First, flush out the 20% ethanol in the column with water, and then use 20 mM Na 3 PO 4Equilibrate the column with a buffer at pH 7.0. After the conductivity on the instrument stabilizes, inject the above antibody supernatant into the column through a 10-ml loop for binding to Protein A at a flow rate of 2 ml / min. After the UV stabilizes, add approximately 0.8 ml of 1 M Tris pH 9.0 buffer (collection volume is approximately 3.2 ml) to the subsequent collection tubes. Then, change the program to elute the antibody bound to the column with 100% 0.1 M Gly pH 3.0, collect the eluate, and then use the method of concentration and buffer exchange to replace the antibody buffer with PBS. The obtained antibody solution can be used directly or aliquoted and stored at -80 °C for later use.

[0128] Example 3: Expression and Conformational Identification of RSV-B-Fm Recombinant Protein

[0129] In this example, the partial RSV-B-Fm recombinant protein (Fm 12-76) expression plasmid constructed in Example 1 was transfected into HEK293T to express the RSV-B-Fm recombinant protein, and the detection antibodies D25 monoclonal antibody and palivizumab prepared in Example 2 were used for detection by ELISA to determine the conformation of the expressed recombinant protein.

[0130] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS. Before transfection, the cell density was made to reach more than 70%. The RSV-B-Fm recombinant protein Fm 12-76 expression plasmid constructed in Example 1 was transfected into HEK293T respectively. After 4-6 hours of 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 the expression of the antigen protein was detected by ELISA using D25 monoclonal antibody and palivizumab respectively. The specific detection methods are as follows:

[0131] (1) Dilute the purified detection antibodies (D25 monoclonal antibody and palivizumab) obtained in Example 2 to 1 μg / ml with ELISA coating buffer (Solarbio, C1050), add 100 μl to each well of a 96-well ELISA plate (Coring, 3590), and place at 4 °C for 12 hours.

[0132] (2) Pour out the coating buffer, add PBS, and wash once; Add 5% skim milk prepared with PBS as a blocking solution to the 96-well plate, 200 μl per well, block, and place at room temperature for 1 hour; After blocking, wash once with PBS solution.

[0133] (3) During the closing period described in step (2), dilute the cell culture supernatant with the closing solution, starting from 5-fold and diluting in 3-fold gradients; then, add 100 μl of the original culture supernatant of each RSV-B recombinant F protein to be tested or the culture supernatant of each dilution of a part of the RSV-B recombinant F proteins (Fm 22, 23, 41, 51, 52, 53, 54, 57) to each well of the ELISA plate. The negative control is to add the closing solution, incubate at 37 °C for 2 hours, and then wash 4 times with PBST;

[0134] (4) Add the Anti-His antibody labeled with HRP (purchased from MBL), incubate at 37 °C for 1.5 hours, and then wash 5 - 6 times with PBST; then, add the TMB chromogenic solution for color development. After reacting for an appropriate time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 reading on the microplate reader.

[0135] The expression results of each RSV-B recombinant F protein in the original supernatant are as Figure 1 and 2 shown. From the Figure 1 and 2 shown results, it can be seen that compared with NC and wild-type RSV-B F protein, the expression of all detected RSV-B-Fm recombinant proteins has been greatly improved; moreover, all detected RSV-B-Fm recombinant proteins can bind to palivizumab (which can recognize both the pre-fusion conformation and the post-fusion conformation of the F protein) and can also bind to the D25 monoclonal antibody (which only recognizes the pre-fusion conformation of the F protein), which indicates that: the RSV-B-Fm recombinant protein of the present invention is in the pre-fusion conformation.

[0136] The expression results of RSV-B recombinant F proteins Fm 22, 23, 41, 51, 52, 53, 54, 57 in the gradient-diluted supernatant are as Figure 3 and 4 shown. From the Figure 3 and 4 shown results, it can be seen that when the cell expression supernatant of RSV-B-Fm 22, 23, 41, 51, 52, 53, 54, 57 is diluted 1000-fold, both the D25 monoclonal antibody and palivizumab can still detect the protein, indicating that: RSV-B-Fm 22, 23, 41, 51, 52, 53, 54, 57 has a high expression level.

[0137] Example 4: Expression, purification and molecular weight identification of RSV-B-Fm recombinant protein

[0138] In this example, the RSV-B-Fm 41, 23, 54 recombinant protein expression plasmids constructed in Example 1 were transfected into HEK293T to express the RSV-B-Fm 41, 23, 54 recombinant proteins, which were purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). At the same time, the target protein was collected and subjected to analytical ultracentrifugation to determine the molecular weight of the expressed recombinant protein.

[0139] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS. Before transfection, the cell density was made to reach more than 70%. HEK293T was transfected with the RSV-B-Fm 41, 23, 54 recombinant protein expression plasmids constructed in Example 1 respectively. After 4-6 hours of transfection, the cell culture medium was replaced with serum-free DMEM and cultured for another 3 days. After collecting the supernatant, DMEM medium was added again and cultured for another 4 days, and the supernatant was collected again. The cell culture supernatants collected twice were mixed and centrifuged at 5000 rpm for 30 minutes. The centrifuged supernatant was filtered through a 0.22 μm filter membrane to bind the target protein to a HisTrap excel column (5 mL, GE Healthcare). Then, the non-specifically bound proteins were eluted with an eluent containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 30 mM imidazole, and the target protein was eluted with an eluent 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 / 300 GL 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 for analytical ultracentrifugation to determine the molecular weight of the expressed recombinant protein.

[0140] The ultraviolet absorption diagram of the molecular sieve chromatography of the recombinant protein RSV-B-Fm 41 is as Figure 5 shown in the left figure, which shows that in the elution peak of the recombinant protein RSV-B-Fm 41, in addition to the target protein peak, there are also a small amount of polymer impurity peaks. The target protein peak was collected for analytical ultracentrifugation, and the result is as Figure 5 shown in the right figure; from Figure 5 the right figure, it can be seen that the measured molecular weight of RSV-B-Fm41 is 170 kDa, which is consistent with the theoretical molecular weight of its trimer, indicating that the expressed RSV-B-Fm 41 recombinant protein is in the trimer form.

[0141] The ultraviolet absorption diagrams of the molecular sieve chromatography of RSV-B-Fm 23 and 54 are respectively as Figure 6 、 7 shown, and fromFigure 6 , 7 It can be seen that in the elution peak of RSV-B-Fm 23, in addition to the target protein peak, there are also a small amount of polymer impurity peaks, while the elution peak of RSV-B-Fm54 is almost a single peak and basically contains no impurity peaks.

[0142] Example 5: Experiment of immunizing mice with RSV-B-Fm recombinant protein

[0143] In this example, the recombinant protein RSV-B-Fm 41 obtained in Example 4 was used to immunize mice; the experimental mice were 4-6-week-old BALB / c mice with an average weight of 15-20 g.

[0144] Specifically, with or without adjuvant, the above-mentioned BALB / c mice were immunized with the recombinant protein RSV-B-Fm41 obtained in Example 4, and the adjuvant was a kind of MF59-like adjuvant - AddaVax; meanwhile, the same dose of normal saline was used for immunization as a negative control.

[0145] The immunization grouping, the immunogens used in each group, the doses of the immunogens used in each group for immunization and the adjuvant conditions are shown in Table 1, and the blank cells indicate "none". The RSV-B-Fm 41 recombinant protein was diluted to the required concentration in normal saline, and the group with adjuvant needed to be further emulsified with the adjuvant in groups, with 6 mice in each group.

[0146] Table 1

[0147]

[0148] Each group of mice was intramuscularly injected with the recombinant protein vaccine or normal saline on days 0 and 14 respectively, and the volume of each immunization was 100 μl. Blood was collected from the tail vein on days 13 and 28 respectively. After the mouse blood was allowed to stand, it was centrifuged at 3000 rpm for 10 minutes to obtain serum. After further inactivating (incubating at 56 °C for 30 minutes), it was aliquoted and stored in a -80 °C refrigerator.

[0149] Example 6: ELISA experiment to detect the specific antibody titer induced by the vaccine

[0150] In this example, the titer of specific IgG antibodies in the serum of mice immunized with the RSV-B-Fm 41 recombinant protein in Example 5 was detected by ELISA experiment.

[0151] Specifically, the following procedure was adopted:

[0152] (1) Dilute the RSV-B-Fm 41 recombinant protein prepared in Example 4 with ELISA coating buffer (Solarbio, C1050) to 3 μg / ml, add 100 μl to each well of a 96-well ELISA plate (Coring, 3590), and place at 4 °C for 12 hours;

[0153] (2) Pour out the coating buffer, add PBS, and wash once; Add 5% skim milk prepared with PBS as a blocking solution to the 96-well plate, 100 μl per well, place at room temperature for 1 hour for blocking; After blocking, wash once with PBS solution;

[0154] (3) During the blocking in step (2), dilute the mouse serum samples with the blocking solution, starting from 10-fold and diluting in a 2-fold gradient; Then, add 100 μl of the immune serum dilution to each well in the ELISA plate, and the negative control is to add the blocking solution, incubate at 37 °C for 2 hours, and then wash 4 times with PBST;

[0155] (4) Add goat anti-mouse secondary antibody conjugated with HRP diluted 1:2000 with the blocking solution (Abcam, ab6789), incubate at 37 °C for 1 hour, and then wash 5 - 6 times with PBST; Add TMB chromogenic solution for color development, after reacting for an appropriate time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 reading on an enzyme-linked immunosorbent assay reader.

[0156] The antibody titer value is defined as the highest dilution factor of the serum when the reaction value is greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.1 times the background value, the titer of this sample is defined as half of the lowest dilution factor, that is, 1:5.

[0157] The results are as Figure 8 shown, Figure 8 The results show that the RSV-B-Fm 41 recombinant protein has good immunogenicity. Among them, after booster immunization with the recombinant protein without adjuvant, the antigen-specific antibody titers induced all reached above 10,000, while after adding AddaVax adjuvant, the antigen-specific antibody titers can be further increased by 10 - 100 times.

[0158] Example 7: Detection of antibody expression and isolation and purification

[0159] According to the methods for constructing the heavy and light chain sequences of antibodies and 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 Microbiol 2,16272(2017); Fabian Sesterhenn., et al. Science 368,eaay5051(2020); Mousa, J., etal. Nat Microbiol 2,16271(2017)), the heavy and light chain expression plasmids of the detection antibodies AM14, MPE8, 101F, and hRSV90 monoclonal antibodies against the RSV F protein were constructed respectively.

[0160] For the specific methods of antibody expression, isolation and purification, refer to Example 2. The finally obtained antibody solution can be used directly or aliquoted and stored in a -80 °C refrigerator for future use.

[0161] Example 8: Expression and conformational identification of RSV-B-Fm recombinant protein

[0162] In this example, all the RSV-B-Fm recombinant protein (Fm 1-76) expression plasmids constructed in Example 1 were used to transfect HEK293T respectively to express the RSV-B-Fm recombinant protein, and the detection antibodies prepared in Examples 2 and 7 were used for detection by ELISA method to determine the expression level and conformation of the expressed recombinant protein. The specific method refers to Example 3.

[0163] The expression results of each RSV-B recombinant F protein in the supernatant stock solution are as Figures 9 - 16 shown, and it can be seen from Figures 9 - 16 that compared with the wild-type RSV-B F protein, the expressions of 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, 71-76 recombinant F proteins were all greatly improved; moreover, these recombinant F proteins can bind both the corresponding monoclonal antibodies against the common epitope II before and after fusion (i.e., palivizumab) and the corresponding monoclonal antibodies against the common epitope IV (i.e., 101F monoclonal antibody), and can also bind the specific epitopes of the pre-fusion conformation such as epitope The corresponding monoclonal antibodies (i.e., D25 monoclonal antibody), the monoclonal antibodies corresponding to the trimer-dependent conformational epitopes (i.e., AM14 monoclonal antibody), the monoclonal antibodies corresponding to epitope V (i.e., hRSV90 monoclonal antibody), or the monoclonal antibodies corresponding to epitope III (i.e., MPE8 monoclonal antibody), which 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 the pre-fusion conformation.

[0164] Example 9: Expression, purification and SDS-PAGE identification of RSV-B-Fm recombinant protein

[0165] In this example, 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, and they were purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The specific operation steps refer to Example 4.

[0166] After gel filtration chromatography, the target protein peak was collected and analyzed by SDS-PAGE. The results are as Figure 17 shown, Figure 17 showing that the RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, 76 recombinant proteins all showed 1 obvious protein band, indicating that they all had high purity, and the molecular weight was between 55KDa - 70Ka, which was consistent with the theoretical molecular weight.

[0167] In addition, since each RSV-B-Fm recombinant F protein is a recombinant protein with a histidine tag, in this example, Western Blot identification was also performed using an anti-histidine tag antibody conjugated with horseradish peroxidase. The results are as Figure 18 shown; Figure 18 showing that the expression of the histidine tag 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 the expectation and was correctly expressed.

[0168] Example 10: Detection of the stability of RSV-B-Fm recombinant protein

[0169] Using the RSV-B-Fm 36, 37, 52, 53, 75, 76 recombinant proteins obtained in Examples 4 and 9, after sterile filtration, they were stored at 30°C. After 4 weeks, by ELISA method, using D25 monoclonal antibody (against epitope ) and palivizumab (targeting epitope II) determine the binding of antigen and antibody through ELISA experiments to determine the stability of the protein during long-term storage. The specific detection method is as follows:

[0170] (1) Dilute the purified antibodies (palivizumab and D25 monoclonal antibody) obtained in Examples 2 and 8 to 1 μg / ml with ELISA coating buffer (Solarbio, C1050) respectively. Add 100 μl to each well of a 96-well ELISA plate (Corning, 3590) and place it at 4°C for 12 hours;

[0171] (2) Pour out the coating buffer, add PBS, and wash once; Add 5% skim milk prepared with PBS as the blocking solution to the 96-well plate, 200 μl per well, block it, and place it at room temperature for 1 hour; After blocking, wash once with PBS solution;

[0172] (3) During the blocking in step (2), dilute the RSV-B-Fm recombinant protein with the blocking solution, starting from 300 ng / ml, and perform serial dilutions at a 3-fold gradient; Then, add 100 μl of the serially diluted RSV-B-Fm recombinant protein to each well of the ELISA plate. The negative control is to add the blocking solution. Incubate at 37°C for 2 hours, and then wash 4 times with PBST;

[0173] (4) Add Anti-His antibody labeled with HRP (purchased from MBL), incubate at 37°C for 1.5 hours, and then wash 5 - 6 times with PBST; Then, add TMB chromogenic solution for color development. After reacting for an appropriate time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 reading on an enzyme-linked immunosorbent assay reader.

[0174] The ELISA test results using D25 monoclonal antibody and palivizumab are shown in Figure 19 and 20 respectively, Figure 19 and 20 show that compared with the protein at the initial week 0, after storing the RSV-B-Fm 36, 37, 52, 53, 75, 76 recombinant proteins at 30°C for 4 weeks, the binding of each protein to D25 antibody and palivizumab did not change significantly, indicating that these RSV-B-Fm recombinant proteins have good stability.

[0175] Example 11: Experiment of immunizing mice with RSV-B-Fm recombinant protein

[0176] Use the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, 76 recombinant proteins obtained in Examples 4 and 10 to immunize mice respectively. The experimental mice are 6 - 8-week-old BALB / c mice with an average weight of 15 - 20 g.

[0177] Specifically, the above RSV-B-Fm recombinant F protein was used to immunize mice, with each immunization dose being 12 μg. The adjuvant was an aluminum hydroxide adjuvant (purchased from Croda) plus a CpG adjuvant (purchased from Invivogen). The vaccine preparation method was as follows: Each RSV-B-Fm recombinant protein was diluted to the required concentration in physiological saline. The aluminum hydroxide adjuvant was first mixed with the antigen protein, and then with the CpG adjuvant. The placebo group was physiological saline. There were 4 BALB / c mice aged 6 - 8 weeks (average body weight 15 - 20 g) in each group.

[0178] Mice in each group were intramuscularly injected with the recombinant protein vaccine or physiological saline on days 0 and 14 respectively, with each immunization volume being 100 μl. Blood was collected from the tail vein on days 13 and 28 respectively. After the mouse blood was allowed to stand, it was centrifuged at 3000 rpm for 10 minutes to obtain serum. After further inactivating (incubating at 56 °C for 30 minutes), it was aliquoted and stored in a -80 °C refrigerator.

[0179] Example 12: Detection of antigen-specific antibody titers induced by the vaccine by ELISA

[0180] In this example, by ELISA, the titers (Log antibody titers) of specific IgG antibodies in the sera of mice after the primary and secondary immunizations with the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, 76 recombinant protein vaccines in Example 11 were detected. The specific method and the definition of antibody titer values were both referred to Example 6.

[0181] The results of the titers of specific IgG antibodies in the sera of mice after the primary and secondary immunizations were respectively as Figure 21 A and Figure 21 B shown, Figure 21 A and 21B showed that the antigen-specific antibody titers induced by all RSV-B-Fm recombinant F protein vaccines after the first immunization reached above 10,000, and the antigen-specific antibody titers induced after the second immunization could be further increased to above 1,000,000, indicating that the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, 76 recombinant F protein vaccines all had excellent immunogenicity.

[0182] Example 13: Evaluation of the protective effect of vaccine immunization against virus challenge

[0183] For each group of mice immunized with the vaccines in Example 11, on the 4th week after the second immunization, they were challenged with the respiratory syncytial virus Long strain by intranasal instillation, and the challenge dose was 10^4 PFU / 50 μl / mouse. On the 5th day after the challenge, the mice were euthanized and the lungs were taken. After weighing and recording the lungs of four mice in each group, DMEM medium was added and then homogenized using a tissue homogenizer. After centrifugation at 5000 g / min for 10 minutes, the lung tissue supernatant was obtained. Through the plaque assay, the virus amount of the respiratory syncytial virus in the lung tissue supernatant sample was detected, and finally the virus copy number per gram of lung tissue was calculated.

[0184] The specific method of the plaque assay is as follows: One day in advance, BHK cells were seeded in a 12-well plate, 1×10 5 cells per well. The starting well was a 10-fold dilution of the tissue stock solution, that is, 50 μl of lung tissue supernatant was added to 450 μl of DMEM medium and mixed evenly, and then serially diluted 6 gradients by 10-fold. The cells to be infected were washed twice with PBS. The diluted lung tissue supernatant sample was taken to infect the cells, 400 μl per well, and incubated at 37 °C for 2 hours. Then the cells were washed with PBS, and then a mixture of 2% carboxymethylcellulose sodium and 2X DMEM in equal volume was added, 1 ml per well. Incubate at 37 °C for 4 days. The cells were washed once with PBS, fixed with methanol added at room temperature for 10 minutes, washed once with PBS, then PBST containing 5% skim milk was added, blocked at 37 °C for 1 hour, palivizumab diluted with 5% skim milk was added, incubated at 37 °C for 1 hour, washed 3 times with PBST (PBS added with 0.05% Tween 20), then horseradish peroxidase-labeled goat anti-human IgG secondary antibody (purchased from Beyotime Biotechnology) was added, incubated at 37 °C for 1 hour, washed 3 times with PBST (PBS added with 0.05% Tween 20), AEC substrate (purchased from BD Biosciences) was added for reaction, and the brown spots were judged as positive plaques, and the PFU value of each sample was counted.

[0185] The results are as Figure 22 shown, Figure 22 showing that after the challenge with the vaccine in the placebo-immunized group, a high titer of respiratory syncytial virus could be detected in the lung tissue, while in all vaccine-immunized groups, no virus was detected. This indicates that after immunization with the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, 76 recombinant protein vaccines, the body can be protected from the infection of the respiratory syncytial virus and a good protective effect is produced.

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A recombinant F protein of respiratory syncytial virus subgroup B, characterized in that, the amino acid sequence of the recombinant F protein of respiratory syncytial virus subgroup B is as shown in SEQ ID NO: 51, 64, 65, 66, 69, 80, 81, 98, 103 or 104.

2. The recombinant F protein of respiratory syncytial virus subgroup B according to claim 1, characterized in that, the recombinant F protein of respiratory syncytial virus subgroup B further comprises a trimer tag; the trimer tag is located at the C-terminus, and its amino acid sequence is as shown in any one of SEQ ID NO: 24-27.

3. The recombinant F protein of respiratory syncytial virus subgroup B according to claim 1 or 2, characterized in that, the recombinant F protein of respiratory syncytial virus subgroup B further comprises a signal peptide; the signal peptide is located at the N-terminus, and its amino acid sequence is as shown in any one of SEQ ID NO: 11-23.

4. A polynucleotide encoding the recombinant F protein of respiratory syncytial virus subgroup B according to any one of claims 1-3.

5. The polynucleotide according to claim 4, characterized in that, the polynucleotide is a DNA molecule or an mRNA molecule.

6. The polynucleotide according to claim 5, characterized in that, the DNA sequence of the DNA molecule is as shown in any one of SEQ ID NO: 127, 140, 141, 142, 145, 156, 157, 174, 179, 180; and / or, the RNA sequence of the mRNA molecule corresponds to the DNA sequence as shown in any one of SEQ ID NO: 127, 140, 141, 142, 145, 156, 157, 174, 179, 180.

7. A nucleic acid construct comprising the polynucleotide according to any one of claims 4-6, and at least one expression regulatory element operably linked to the polynucleotide.

8. An expression vector comprising the nucleic acid construct according to claim 7.

9. A host cell transformed or transfected with the polynucleotide according to any one of claims 4-6, the nucleic acid construct according to claim 7, or the expression vector according to claim 8.

10. The host cell according to claim 9, characterized in that, the host cell is a mammalian cell, an insect cell, a yeast cell or a bacterial cell.

11. The host cell according to claim 10, characterized in that, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell; the bacterial cell is an Escherichia coli cell.

12. A recombinant F protein trimer of respiratory syncytial virus subgroup B, which is formed by polymerization of three recombinant F proteins of respiratory syncytial virus subgroup B as described in claim 2.

13. Use of the respiratory syncytial virus subgroup B recombinant F protein according to any one of claims 1 - 3, the polynucleotide according to any one of claims 4 - 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, the host cell according to any one of claims 9 - 11, or the respiratory syncytial virus subgroup B recombinant F protein trimer according to claim 12 in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection.

14. A vaccine or immunogenic composition comprising the respiratory syncytial virus subgroup B recombinant F protein according to any one of claims 1 - 3, the polynucleotide according to any one of claims 4 - 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, the host cell according to any one of claims 9 - 11, or the respiratory syncytial virus subgroup B recombinant F protein trimer according to claim 12, and a physiologically acceptable vehicle, adjuvant, excipient, carrier, and / or diluent.

15. The vaccine or immunogenic composition according to claim 14, which is a respiratory syncytial virus recombinant protein vaccine, comprising the respiratory syncytial virus subgroup B recombinant F protein according to any one of claims 1 - 3 or the respiratory syncytial virus subgroup B recombinant F protein trimer according to claim 12 and an adjuvant.

16. The immunogenic composition according to claim 15, wherein, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59 - like adjuvant, and AS series - like adjuvants.

17. The vaccine or immunogenic composition according to claim 14, which is a respiratory syncytial virus DNA vaccine, the DNA vaccine comprising: (i) a eukaryotic expression vector; and (ii) a DNA sequence encoding the respiratory syncytial virus subgroup B recombinant F protein according to any one of claims 1 - 3, which is constructed into the eukaryotic expression vector.

18. The vaccine or immunogenic composition according to claim 17, wherein, the DNA sequence is as shown in any one of SEQ ID NO: 127, 140, 141, 142, 145, 156, 157, 174, 179, 180; and / or, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS, and pcDNA series vectors.

19. The vaccine or immunogenic composition according to claim 14, which is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising: (I) an mRNA sequence encoding the respiratory syncytial virus subgroup B recombinant F protein according to any one of claims 1 - 3; and (II) a lipid nanoparticle.

20. The vaccine or immunogenic composition according to claim 19, wherein, the mRNA sequence corresponds to the DNA sequence as shown in any one of SEQ ID NO: 127, 140, 141, 142, 145, 156, 157, 174, 179, 180.

21. The vaccine or immunogenic composition according to claim 14, which is a respiratory syncytial virus viral vector vaccine, and which comprises: (1) a viral backbone vector; and (2) a DNA sequence encoding the recombinant respiratory syncytial virus subgroup B F protein as described in any one of claims 1-3, which is constructed into the viral backbone vector.

22. The vaccine or immunogenic composition according to claim 21, wherein the DNA sequence is as shown in any one of SEQ ID NO: 127, 140, 141, 142, 145, 156, 157, 174, 179, 180; and / or, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

23. The vaccine or immunogenic composition according to any one of claims 14-22, wherein the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation.

24. The vaccine or immunogenic composition according to claim 23, wherein the nasal spray is selected from aerosols, sprays and powder aerosols; the oral preparation is selected from tablets, powders, pills, powders, granules, soft capsules, hard capsules, film-coated tablets and ointments; the parenteral preparation is a transdermal agent, ointment, plaster, external liquid preparation, injectable or pushable preparation.

25. The method for preparing the recombinant respiratory syncytial virus subgroup B F protein according to any one of claims 1-3, wherein the preparation method comprises: adding a nucleotide sequence encoding a signal peptide to the 5' end and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon to the 3' end of the codon-optimized nucleotide sequence encoding the recombinant respiratory syncytial virus subgroup B F protein as described in any one of claims 1-3, performing cloning and expression, screening for correct recombinants, and then transfecting the expression system cells for expression, collecting the cell culture supernatant, and isolating the recombinant respiratory syncytial virus subgroup B F protein therefrom.

26. The preparation method according to claim 25, wherein the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells.

27. The preparation method according to claim 26, wherein the mammalian cells are 293T cells, 293F cells, or CHO cells; the bacterial cells are Escherichia coli cells.

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