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

Abstract

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

Description

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on September 29, 2022, with application number 202211199603.X, and invention name "A recombinant fusion protein of respiratory syncytial virus in a pre-fusion conformation, its preparation method and use", the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of vaccine technology, and in particular to a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, a stabilized trimer formed thereby, an immunogenic composition containing the same, and a preparation method and use thereof. Background Art

[0004] Respiratory syncytial virus (RSV) is the most common pathogen causing acute respiratory tract infections in infants. Bronchiolitis caused by RSV infection is one of the main causes of hospitalization in children under 2 years old and an important factor leading to rising 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 old with severe RSV infection worldwide reached 34.6 million, of which 3 million were infected in China alone. 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 reveal that the annual incidence of RSV infection is between 2% and 10%. Large-scale epidemiological studies have revealed that the hospitalization and mortality rates caused by RSV in the elderly population are comparable to those of influenza.

[0005] RSV has only a single serotype with two major antigenic subgroups, A and B. Strains of the two subtypes usually co-circulate, but only one of the subtypes usually dominates during epidemics. In temperate regions including my country, RSV infection shows obvious seasonality, with an onset in late autumn to early winter, peaking between mid-December and early February, and beginning to decline in late spring. There is an urgent need for safe, effective and inexpensive preventive treatments for RSV virus infection worldwide.

[0006] RSV belongs to the genus Pneumovirus of the family Paramyxoviridae. It is a polymorphic enveloped virus with a diameter of about 120–300 nm and a non-segmented negative-sense single-stranded RNA (15–16 kb) encoding 11 proteins. The viral envelope contains three proteins, namely the attachment glycoprotein (G), the fusion (F) glycoprotein, and the small hydrophobic (SH) protein. The G protein plays a role in host cell attachment, and the F protein is responsible for fusion and cell entry, while the SH protein is not required for both processes. The two surface glycoproteins of RSV are the main neutralizing antigens: the G protein has a high sequence diversity and determines the antigenic subtype of the virus (A and B); 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 highly conserved protein sequence, the RSV F protein is the target of neutralizing antibodies and the main antigen for vaccine development. The F protein belongs to a type I transmembrane protein. The F protein is first synthesized as a 574-amino acid precursor protein F0, which has 5-6 post-translationally modified N-linked glycosylation modifications on the F0 protein. In order to become a functional fusion protein, F0 is treated by cellular furin-like proteases at two polybasic sites in the trans-Golgi apparatus to produce F1 (amino acids 137-574), F2 (amino acids 26-109) and Pep27 polypeptides, and finally form two fragments F1 and F2 connected by disulfide bonds, with sizes of 55kD and 15kD, respectively. The homologous F protein trimer contains two conformations, pre-fusion and post-fusion. The pre-fusion F protein is a metastable structure. When the virus fuses with the cell, the pre-fusion F is transformed into a stable post-fusion F protein, and this process can also be transformed spontaneously. Since the highly active neutralizing antibody epitopes 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 the development of RSV vaccines. To date, there is still no RSV vaccine on the market. 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 referred to 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 above-mentioned polynucleotide, nucleic acid construct or expression vector, a stabilized trimer formed by the recombinant F protein, an immunogenic composition comprising any of the foregoing, and use thereof 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 the first aspect, the present invention provides a recombinant F protein of respiratory syncytial virus subtype B (RSV-B), which is a recombinant F protein based on respiratory syncytial virus subtype B, also referred to herein as RSV-B recombinant protein or RSV-B recombinant F protein, the RSV-B recombinant F protein comprising RSV-B-F1 peptide segment and RSV-B-F2 peptide segment, wherein the RSV-B-F1 peptide segment and / or RSV-B-F2 peptide segment contain at least one mutation to proline relative to the corresponding peptide segment of wild-type RSV-B F protein.

[0010] In a specific embodiment, the RSV-B-F1 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-BF protein as shown in SEQ ID NO:1, and the RSV-B-F2 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 as shown in SEQ ID NO:1, and the RSV-B-F1 peptide segment and / or RSV-B-F2 peptide segment contain 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 selected from the following mutations in the amino acid sequence of the wild-type RSV-BF protein as 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 as shown in SEQ ID NO: 29;

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

[0016] Q279P; for example, in the RSV-B recombinant F protein Fm 3 as 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 mutation or combination of mutations may be replaced by a K65P mutation (e.g., RSV-B recombinant F protein Fm 24 as shown in SEQ ID NO: 52) or a D73P mutation (e.g., RSV-B recombinant F protein Fm25 as shown in SEQ ID NO: 53);

[0038] Optionally, the L141P mutation in the above mutation or combination of mutations may be replaced by the L138P mutation or the G139P mutation;

[0039] Optionally, the above mutation or combination of mutations further includes 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 above mutations further introduce an N216P mutation, for example, in RSV-B recombinant F protein Fm 26-34 whose amino acid sequence is as shown in any one of SEQ ID NOs: 54-62; in other embodiments, the above mutations further introduce a Q210P mutation, for example, in RSV-B recombinant F protein Fm35 whose amino acid sequence is as shown in SEQ ID NO: 63; in other embodiments, the above mutations further introduce an I214P mutation, for example, in RSV-B recombinant F protein Fm 36 whose amino acid sequence is as shown in SEQ ID NO: 64; in other embodiments, the above mutations further introduce an S215P mutation, for example, in RSV-B recombinant F protein Fm 37-38 whose amino acid sequence is as shown in SEQ ID NOs: 65-66; in other embodiments, the above mutations further introduce an E161P mutation, for example, in RSV-B recombinant F protein Fm 39-55 whose amino acid sequence is as shown in any one of SEQ ID NOs: 67-83.

[0041] In some embodiments, the RSV-B-F1 peptide segment and the RSV-B-F2 peptide segment are directly linked, for example, in RSV-B recombinant F protein Fm 74-76 (as shown in SEQ ID NO: 102-104).

[0042] In other embodiments, the RSV-B-F1 peptide segment and the RSV-B-F2 peptide segment are connected via a connecting bridge;

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

[0044] (i) (GS) m connecting bridge, wherein m = 1-5, preferably 1-3; optionally, comprising one or more amino acid mutations; for example, as shown in SEQ ID NO: 9, such as in RSV-B recombinant F protein Fm 69-71 (such as shown in SEQ ID NO: 97-99), or as shown in SEQ ID NO: 10, such as in RSV-B recombinant F protein Fm 72-73 (such as shown in SEQ ID NO: 100-101));

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

[0046] (iii) a linker sequence between the wild-type F1 and F2 peptide segments and F0 itself, optionally, a sequence as shown in SEQ ID NO: 7 (e.g., in RSV-B recombinant F protein Fm 63-65 (e.g., SEQ ID NO: 91-93));

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

[0048] Preferably, the connecting bridge comprises or consists of an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NOs: 2-10.

[0049] 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: 29-104, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in any one of SEQ ID NOs: 29-104, and having the same or substantially the same immunogenicity as the amino acid sequence.

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

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

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

[0053] Furthermore, 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, which may 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 one of SEQ ID NOs: 105-180, or consists of it.

[0058] In other embodiments, the polynucleotide is an mRNA molecule, preferably, the mRNA molecule comprises or consists of an RNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 105-180.

[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 control 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, wherein the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect is transformed or transfected;

[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 subtype B (RSV-B) recombinant F protein trimer, which is polymerized by three RSV-B recombinant F proteins as described in the first aspect above.

[0066] In the seventh aspect, the present invention provides the use of the RSV-B recombinant F protein as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the RSV-B recombinant F protein trimer as described in the sixth aspect 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, comprising the RSV-B recombinant F protein as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV-B recombinant F protein trimer as described in the sixth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0068] In some preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, which includes 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 adjuvant.

[0070] In other preferred 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 constructed into the eukaryotic expression vector, preferably a DNA sequence as shown in any one of SEQ ID NOs: 105-180;

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

[0074] In other preferred 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 a DNA sequence as shown in any one of SEQ ID NOs: 105-180; and

[0076] (II) Lipid nanoparticles.

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

[0078] (1) viral backbone vectors; and

[0079] (2) a DNA sequence encoding the RSV-B recombinant F protein as described in the first aspect constructed into the viral backbone vector, preferably a DNA sequence as shown in any one of SEQ ID NOs: 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, the vaccine or immunogenic composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;

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

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

[0084] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a 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] 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 as described in the first aspect above, and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon are added to the 3' end, and cloned expression is performed, the correct recombinant is screened, and then it is transfected into expression system cells for expression, the cell culture supernatant is collected, and the RSV-B recombinant F protein is isolated 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 natural RSV-B virus F protein antigen is recombinantly expressed in vitro, the pre-fusion conformation is unstable and the F antigen protein in the pre-fusion conformation cannot be obtained. In the present invention, one or more mutations to proline are introduced into the F1 and / or F2 peptide segments of the respiratory syncytial virus type B F protein to form a stable RSV-B recombinant F protein in the pre-fusion conformation of the present invention; experiments show that the formed RSV-B recombinant F protein contains at least one specific epitope of the pre-fusion F protein, and its expression is stable, 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 boosting immunization with recombinant protein without adding adjuvant, the induced antigen-specific antibody titer reaches more than 10,000, and after adding AddaVax adjuvant, the antigen-specific antibody titer can be further increased by 10-100 times), which is of 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 exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0091] Figure 1 In Example 3, the expression of RSV-B recombinant F protein in the supernatant stock solution was detected by ELISA using D25 monoclonal antibody, wherein the horizontal axis is the experimental group (ie, the detected RSV-B recombinant F protein) and the vertical axis is the OD450 nm absorbance.

[0092] Figure 2 In Example 3, the expression of RSV-B recombinant F protein in the supernatant stock solution was detected by ELISA using palivizumab, wherein the horizontal axis represents the experimental group (i.e., the detected RSV-B recombinant F protein) and the vertical axis represents the OD450nm absorbance value.

[0093] Figure 3In Example 3, the expression of some RSV-B recombinant F proteins in the diluted supernatant was detected by ELISA using D25 monoclonal antibody, wherein the abscissa is the Log10 value of the dilution multiple of the cell supernatant, the ordinate is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the detected RSV-B recombinant F protein).

[0094] Figure 4 In Example 3, palivizumab was used to detect the expression of some RSV-B recombinant F proteins in the diluted supernatant by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450nm absorbance value, and the legend on the right shows the experimental group (i.e., the detected RSV-B recombinant F protein).

[0095] Figure 5 The graphs showing the results of purification of the expression supernatant of RSV-B recombinant F protein Fm41 by molecular sieve chromatography and analytical ultracentrifugation of the purified protein in Example 4 are shown, wherein the left graph is an ultraviolet absorption graph of molecular sieve chromatography of the expression supernatant of RSV-B recombinant F protein Fm41 during purification, and the right graph is the result of analytical ultracentrifugation of the purified RSV-B recombinant F protein Fm41.

[0096] Figure 6 The ultraviolet absorption graph of the expression supernatant of RSV-B recombinant F protein Fm23 in Example 4 is shown when it is purified by molecular sieve chromatography.

[0097] Figure 7 The ultraviolet absorption graph of the expression supernatant of RSV-B recombinant F protein Fm54 in Example 4 is shown when it is purified by molecular sieve chromatography.

[0098] Figure 8 In Example 6, the titer level of specific antibody IgG in the serum of mice recorded in Example 5 after primary and booster immunization of mice using RSV-B recombinant F protein Fm 41 as an immunogen was detected by ELISA experiment, wherein the horizontal axis is the grouping in Table 1 and the vertical axis is the antibody titer.

[0099] Fig. 9 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0100] Fig.10 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0101] Fig.11 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0102] Fig.12 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0103] Fig.13 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0104] Fig.14In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0105] Fig.15 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0106] Fig.16 In Example 8, D25, AM14, palivizumab, 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 with the above antibodies by ELISA, wherein the horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, the vertical axis is the OD450 nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-B recombinant F protein detected).

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

[0108] Fig.18 The Western Blot test results of the isolated and purified RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 recombinant F proteins in Example 9 are shown.

[0109] Fig.19 In Example 10, D25 monoclonal antibody was used to detect the binding of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins with antibodies before and after storage at 30°C for 4 weeks by ELISA, wherein the abscissa is the protein concentration and the ordinate is the OD450 nm absorbance.

[0110] Fig. 20In Example 10, palivizumab was used to detect the binding of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins with antibodies before and after storage at 30°C for 4 weeks by ELISA, wherein the abscissa is the protein concentration and the ordinate is the OD450 nm absorbance.

[0111] Fig.21 In Example 12, an ELISA experiment was performed to detect the titer levels of specific binding antibody IgG in the serum of mice recorded in Example 11 after the mice were given primary immunization (A) and booster immunization (B) using RSV-B-Fm23, 36, 37, 38, 41, 52, 53, 54, 70, 75, and 76 recombinant F proteins as immunogens, respectively, wherein the horizontal axis represents the vaccine grouping and the vertical axis represents the antibody titer.

[0112] Fig. 22 In Example 13, the viral load of the lung tissue of mice immunized with the vaccine described in Example 11 and challenged with respiratory syncytial virus by nasal drops was detected by plaque assay, wherein the horizontal axis represents the vaccine grouping and the vertical axis represents the viral load. DETAILED DESCRIPTION

[0113] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0114] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0115] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0116] Example 1: Construction of the expression plasmid for the 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 were shown in SEQ ID NOs: 29-104, respectively.

[0118] According to the preference of mammalian cell codons, the nucleic acid sequence encoding the above-mentioned RSV-B recombinant F protein of the present invention, RSV-B-Fm 1-76, was optimized to obtain optimized nucleic acid coding sequences, as shown in SEQ ID NOs: 105-180, respectively; an EcoRI restriction site sequence, a Kozak sequence, and a nucleic acid coding sequence of a signal peptide (as shown in SEQ ID NOs: 11-23) were added to the 5' end of the nucleic acid coding sequence as shown in SEQ ID NOs: 105-180, and a nucleic acid coding sequence of a trimer tag (as shown in SEQID NOs: 24-27), a nucleic acid coding sequence of a His tag (as shown in SEQ ID NO: 28), a stop codon and an XhoI restriction site sequence were added to the 3' end, and then GeneScript Biotech Co., Ltd. was commissioned to perform gene synthesis, and then it was connected to the pCAGGS vector through the two restriction sites of EcoRI and XhoI to obtain an expression plasmid expressing the recombinant protein RSV-B-Fm 1-76.

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

[0120] Fm 1-37 (amino acid sequences are shown in SEQ ID NOs: 29-65, respectively, and optimized nucleic acid coding sequences are shown in SEQ ID NOs: 105-141, respectively) and Fm 39-76 (amino acid sequences are shown in SEQ ID NOs: 67-104, respectively, and optimized nucleic acid coding sequences are shown in SEQ ID NOs: 143-180, respectively) all use the signal peptide shown in SEQ ID NO: 14 and the trimer tag shown in SEQ ID NO: 24;

[0121] Fm 38 (amino acid sequence shown in SEQ ID NO:66, optimized nucleic acid coding sequence 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: Expression and purification of detection antibodies

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

[0124] Antibody expression

[0125] 14-16 hours before transfection, the cells with higher density of 293T cells were divided into plates (e.g., a 10cm culture dish with 100% 293T cells was passaged at 1:3). After 14-16 hours, the cell density reached more than 70% and transfection could be performed. During transfection, the heavy chain and light chain plasmids of the antibody were co-transfected into 293T cells at a ratio of 2:3. After 4-6 hours of transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium for continued culture. The cell supernatants were collected on the 3rd and 7th days after transfection, and the cell debris was removed by centrifugation. The antibody supernatants obtained twice were mixed for subsequent antibody protein purification.

[0126] Antibody purification

[0127] A Protein A (5 ml) HP affinity column (GE) was connected to an AKTA Purifier / Explorer / FPLC / START (GE). The operation process on the instrument was as follows: first, the 20% ethanol in the column was flushed out with water, and then the column was equilibrated with a 20 mM Na3PO4, pH 7.0 buffer. After the conductivity on the instrument was stable, the above antibody supernatant was injected through a 10 ml loop loading method to bind to Protein A, with a flow rate of 2 ml / min. After the UV was stable, about 0.8 ml of 1 M Tris pH 9.0 buffer was added to the subsequent collection tube (collection volume was about 3.2 ml), and then the program was changed to 100% 0.1 M Gly pH 3.0 to elute the antibody bound to the column, the eluate was collected, and then the antibody buffer was replaced with PBS by the concentration and liquid replacement method. The obtained antibody solution can be used directly or stored in a -80°C refrigerator for later use.

[0128] Example 3: Expression and conformation identification of RSV-B-Fm recombinant protein

[0129] In this example, HEK293T was transfected with a partial RSV-B-Fm recombinant protein (Fm 12-76) expression plasmid constructed in Example 1 to express 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, and before transfection, the cell density reached more than 70%; HEK293T was transfected with the RSV-B-Fm recombinant protein Fm 12-76 expression plasmid constructed in Example 1. After 4-6 hours of transfection, the cell culture medium was replaced with serum-free DMEM, and the culture was continued for 3 days. The cell culture supernatant was collected, and the antigen protein expression was detected by ELISA method using D25 monoclonal antibody and palivizumab, respectively. The specific detection method is as follows:

[0131] (1) The purified detection antibodies (D25 monoclonal antibody and palivizumab) obtained in Example 2 were diluted to 1 μg / ml with ELISA coating solution (Solebol, C1050), 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590), and placed at 4°C for 12 hours;

[0132] (2) Pour off the coating solution, add PBS, and wash once; add 5% skim milk prepared in PBS as blocking solution into the 96-well plate, 200 μl per well, block, and leave at room temperature for 1 hour; after blocking, wash once with PBS solution;

[0133] (3) During the blocking period of step (2), the cell culture supernatant was diluted with blocking solution, starting from 5 times and then diluted in a 3-fold gradient; then, 100 μl of the culture supernatant of each RSV-B recombinant F protein to be tested or the culture supernatant of each dilution of a portion of RSV-B recombinant F protein (Fm 22, 23, 41, 51, 52, 53, 54, 57) was added to each well of the ELISA plate. The negative control was added with blocking solution, incubated at 37 degrees for 2 hours, and then washed 4 times with PBST;

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

[0135] The expression results of each RSV-B recombinant F protein in the supernatant stock solution are as follows Figure 1 and 2 As shown, from Figure 1 and 2The results show that compared with NC and wild-type RSV-B F proteins, the expression of all RSV-B-Fm recombinant proteins tested was greatly improved; moreover, all RSV-B-Fm recombinant proteins tested can bind to both palivizumab (which recognizes both the F protein in the pre-fusion conformation and the F protein in the post-fusion conformation) and D25 monoclonal antibody (which only recognizes the F protein in the pre-fusion conformation), 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 protein Fm 22, 23, 41, 51, 52, 53, 54, 57 in the gradient dilution supernatant are as follows Figure 3 and 4 As shown, from Figure 3 and 4 The results show that when the expression supernatant of RSV-B-Fm 22, 23, 41, 51, 52, 53, 54, and 57 cells was diluted 1000 times, the protein could still be detected by D25 monoclonal antibody or palivizumab, indicating that RSV-B-Fm 22, 23, 41, 51, 52, 53, 54, and 57 had a higher 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 used to transfect HEK293T to express 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"). The target protein was collected and then 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, and before transfection, the cell density reached more than 70%; HEK293T was transfected with RSV-B-Fm 41, 23, and 54 recombinant protein expression plasmids constructed in Example 1 respectively; 4-6 hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the culture was continued for 3 days. After collecting the supernatant, DMEM medium was added again, and the culture was continued for 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 allow the target protein to bind to the HisTrap excel column (5 mL, GE Healthcare), and then the non-specifically bound protein was eluted with an eluent containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 30 mM imidazole, and then the target protein was eluted with an eluent containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 400 mM imidazole; the fractions containing the target protein were collected, concentrated, and then molecular sieve chromatography was performed (Superdex 200 Increase 10 / 300 GL or Superdex 200 Hiload 16 / 60 GE Healthcare) to obtain 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 UV absorption graph of the molecular sieve chromatography of the recombinant protein RSV-B-Fm 41 is shown in Figure 5 As shown in the left figure, it shows that the elution peak of the recombinant protein RSV-B-Fm 41 contains a small amount of polymer impurity peak in addition to the target protein peak; the target protein peak is collected for analytical ultracentrifugation, and the results are as follows Figure 5 As shown in the figure on the right; Figure 5 As can be seen from the right figure, the measured molecular weight of RSV-B-Fm41 is 170 kDa, which is consistent with its theoretical molecular weight of trimer, indicating that the expressed RSV-B-Fm 41 recombinant protein is in the form of a trimer.

[0141] The UV absorption patterns of RSV-B-Fm 23 and 54 by molecular sieve chromatography are shown in Figure 6 , 7 As shown by Figure 6 , 7 It can be seen that the elution peak of RSV-B-Fm 23 contains not only the target protein peak but also a small amount of polymer impurity peaks, while the elution peak of RSV-B-Fm54 is almost a single peak and basically contains no impurity peaks.

[0142] Example 5: RSV-B-Fm recombinant protein immunization mouse experiment

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

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

[0145] The immunization groups, the immunogens used in each group, the immunogen doses used in each group, and the adjuvants used are shown in Table 1. Blank cells represent “none.” RSV-B-Fm 41 recombinant protein was diluted to the required concentration in physiological saline. The adjuvant-added group was further emulsified with the adjuvant, with 6 mice in each group.

[0146] Table 1

[0147]

[0148] Each group of mice was injected intramuscularly with recombinant protein vaccine or normal saline on days 0 and 14, with each immunization volume of 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, which was further inactivated (incubated at 56°C for 30 minutes) and then stored in a -80°C refrigerator.

[0149] Example 6: ELISA test to detect vaccine-induced specific antibody titers

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

[0151] Specifically, the following procedure is adopted:

[0152] (1) The RSV-B-Fm 41 recombinant protein prepared in Example 4 was diluted to 3 μg / ml with ELISA coating solution (Solabo, C1050), 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590), and the plate was placed at 4°C for 12 hours;

[0153] (2) Pour off the coating solution, add PBS, and wash once; add 5% skim milk prepared in PBS as blocking solution to the 96-well plate, 100 μl per well, and leave at room temperature for 1 hour for blocking; after blocking, wash once with PBS solution;

[0154] (3) During the blocking period of step (2), the mouse serum samples were diluted with blocking solution, starting from 10 times and diluted in a 2-fold gradient; then, 100 μl of immune serum dilution solution was added to each well of the ELISA plate, and the negative control was added with blocking solution, incubated at 37 degrees for 2 hours, and then washed 4 times with PBST;

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

[0156] The antibody titer value is defined as the highest serum dilution factor with a reaction value 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 the sample is defined as half of the lowest dilution factor, i.e. 1:5.

[0157] The results are as follows Figure 8 As shown, Figure 8 The results showed that RSV-B-Fm 41 recombinant protein has good immunogenicity. After booster immunization with the recombinant protein without adding adjuvant, the induced antigen-specific antibody titer reached more than 10,000. After adding AddaVax adjuvant, the antigen-specific antibody titer could be further increased by 10-100 times.

[0158] Example 7: Expression and purification of detection antibodies

[0159] According to the construction methods of antibody heavy chain and light chain sequences and 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., et al. Nat Microbiol 2, 16271(2017)), the heavy chain and light chain expression plasmids of RSV F protein detection antibodies - AM14, MPE8, 101F and hRSV90 monoclonal antibodies were constructed respectively.

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

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

[0162] In this example, all RSV-B-Fm recombinant protein (Fm 1-76) expression plasmids constructed in Example 1 were used to transfect HEK293T, thereby expressing RSV-B-Fm recombinant protein, and the detection antibodies prepared in Examples 2 and 7 were used to detect by ELISA method to determine the expression amount and conformation of the expressed recombinant protein. For specific methods, refer to Example 3.

[0163] The expression results of each RSV-B recombinant F protein in the supernatant stock solution are as follows Figure 9-16 As shown by Figure 9-16 It can be seen that compared with the wild-type RSV-B F protein, the expression 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, and 71-76 recombinant F proteins were greatly improved; moreover, these recombinant F proteins can bind to the corresponding monoclonal antibody of the shared epitope II before and after fusion (i.e., palivizumab) and the corresponding monoclonal antibody of the shared epitope IV (i.e., 101F monoclonal antibody), and can also bind to the specific epitope of the prefusion conformation, such as the epitope. The corresponding monoclonal antibody (i.e., D25 monoclonal antibody), the corresponding monoclonal antibody of the trimer-dependent spatial conformation epitope (i.e., AM14 monoclonal antibody), the corresponding monoclonal antibody of epitope V (i.e., hRSV90 monoclonal antibody), or the corresponding monoclonal antibody of 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 protein of the present invention is a pre-fusion conformation.

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

[0165] In this example, the RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, 76 recombinant protein expression plasmids constructed in Example 1 were used to transfect HEK293T to express RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, 76 recombinant proteins, and purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The specific operating steps are shown in Example 4.

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

[0167] In addition, since all RSV-B-Fm recombinant F proteins are recombinant proteins with histidine tags, this example also uses an anti-histidine tag antibody coupled with horseradish peroxidase for Western Blot identification. The results are as follows: Fig.18 As shown; Fig.18 It was shown that the expression of histidine tags was detected in each RSV-B-Fm recombinant F protein collection fluid, indicating that the expression of these RSV-B-Fm recombinant F proteins was consistent with expectations and was correctly expressed.

[0168] Example 10: Stability test of RSV-B-Fm recombinant protein

[0169] The RSV-B-Fm 36, 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 D25 monoclonal antibody (anti-epithelial marker) was used for ELISA. ) and palivizumab (targeting epitope II) were used to determine the binding of antigen and antibody by ELISA experiment to determine the stability of protein in long-term storage. The specific detection method is as follows:

[0170] (1) The purified antibodies (palivizumab and D25 monoclonal antibody) obtained in Examples 2 and 8 were diluted to 1 μg / ml with ELISA coating solution (Solebol, C1050), 100 μl was added to each well of a 96-well ELISA plate (Corning, 3590), and the plate was placed at 4°C for 12 hours;

[0171] (2) Pour off the coating solution, add PBS, and wash once; add 5% skim milk prepared in PBS as blocking solution into the 96-well plate, 200 μl per well, block, and leave at room temperature for 1 hour; after blocking, wash once with PBS solution;

[0172] (3) During the blocking period of step (2), the RSV-B-Fm recombinant protein was diluted with blocking solution, starting from 300 ng / ml and diluted in a 3-fold gradient; then, 100 μl of the gradient diluted RSV-B-Fm recombinant protein was added to each well of the ELISA plate. The negative control was to add blocking solution, incubate at 37 degrees for 2 hours, and then wash 4 times with PBST;

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

[0174] The ELISA test results using D25 monoclonal antibody and palivizumab were as follows: Fig.19 and 20 As shown, Fig.19 and 20 The results showed that compared with the initial proteins at week 0, after storage at 30°C for 4 weeks, the binding of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant proteins to D25 antibody and palivizumab did not change significantly, indicating that these RSV-B-Fm recombinant proteins have good stability.

[0175] Example 11: RSV-B-Fm recombinant protein immunization mouse experiment

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

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

[0178] Each group of mice was injected intramuscularly with recombinant protein vaccine or normal saline on days 0 and 14, with each immunization volume of 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, which was further inactivated (incubated at 56°C for 30 minutes) and then stored in a -80°C refrigerator.

[0179] Example 12: ELISA test to detect vaccine-induced antigen-specific antibody titers

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

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

[0182] Example 13: Evaluation of vaccine immune challenge protection effect

[0183] For each vaccine-immunized mouse in Example 11, the respiratory syncytial virus Long strain was challenged by nasal drops in the 4th week after the secondary immunization, and the challenge dose was 10^4PFU / 50μl / mouse. On the 5th day after the challenge, the mice were euthanized and the lungs were taken. After the lungs of four mice in each group were weighed and recorded, DMEM medium was added and then homogenized using a tissue homogenizer, and the lung tissue supernatant was obtained after centrifugation at 5000g / min for 10 minutes. The amount of respiratory syncytial virus in the lung tissue supernatant sample was detected by plaque assay, and the number of viral copies per gram of lung tissue was finally calculated.

[0184] The specific method of the plaque assay is as follows: use a 12-well plate to plate BHK cells one day in advance, with 1*10 5cells, the starting well is a 10-fold dilution of the tissue stock solution, that is, 50μl of lung tissue supernatant is added to 450μl DMEM medium and mixed, and then diluted 6 times in a 10-fold gradient. Wash the cells to be infected twice with PBS, take the diluted lung tissue supernatant sample to infect the cells, 400μl per well, incubate at 37℃ for 2 hours, then wash the cells with PBS, add an equal volume of 2% sodium carboxymethyl cellulose and 2XDMEM, add 1ml per well. Incubate at 37℃ for 4 days. The cells were washed once with PBS, fixed with methanol at room temperature for 10 minutes, washed once with PBS, and then added with PBST containing 5% skim milk, blocked at 37°C for 1 hour, and then added with palivizumab diluted with 5% skim milk, incubated at 37°C for 1 hour, washed three times with PBST (PBS with 0.05% Tween20), and then added with horseradish peroxidase-labeled goat anti-human IgG secondary antibody (purchased from Biyuntian Biotechnology), incubated at 37°C for 1 hour, washed three times with PBST (PBS with 0.05% Tween20), and AEC substrate (purchased from BD Biosciences) was added for reaction. Brown spots were determined as positive spots, and the PFU value of each sample was counted.

[0185] The results are as follows Fig. 22 As shown, Fig. 22 The results showed that high titers of respiratory syncytial virus were detected in the lung tissue of the placebo immunization group after vaccine challenge, while no virus was detected in all vaccine immunization groups. This indicates that RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant protein vaccines can protect the body from respiratory syncytial virus infection after immunization, and have a good protective effect.

[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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present invention.

Claims

1. A recombinant F protein of respiratory syncytial virus subtype B (RSV-B), characterized in that The RSV-B recombinant F protein comprises an RSV-B-F1 peptide segment and an RSV-B-F2 peptide segment, wherein the RSV-B-F1 peptide segment and / or the RSV-B-F2 peptide segment comprise at least one mutation to proline relative to the corresponding peptide segment of the wild-type RSV-B F protein.

2. The RSV-B recombinant F protein according to claim 1, characterized in that The RSV-B-F1 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 as shown in SEQ ID NO:1, and the RSV-B-F2 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 as shown in SEQ ID NO:1, and the RSV-B-F1 peptide segment and / or RSV-B-F2 peptide segment contain one or more mutations to proline relative to the corresponding peptide segment of the wild-type RSV-B F protein.

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

4. The RSV-B recombinant F protein according to any one of claims 1 to 3, characterized in that The RSV-B-F1 peptide segment and the RSV-B-F2 peptide segment are directly connected or connected via a connecting bridge; Preferably, the connecting bridge is selected from: (i) (GS)m connecting bridge, wherein m=1-5, preferably 1-3; optionally, comprising one or more amino acid mutations; (ii) (GGGGS)n linker, wherein n=1-5, preferably 1-3; optionally, comprising one or more amino acid mutations; (iii) the linker sequence between the wild-type F1 and F2 peptides and F0 itself; (iv) a sequence obtained by mutating the furin cleavage site on the connecting bridge (iii); Further preferably, the connecting bridge comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-10.

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 following: SEQ ID NO: 29-104, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in any one of SEQ ID NO: 29-104, and having the same or substantially the same immunogenicity as the amino acid sequence.

6. The RSV-B recombinant F protein according to any one of claims 1 to 5, characterized in that The recombinant F protein also includes a trimer tag; Preferably, the trimer tag is located at the C-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO: 24-27.

7. The RSV-B recombinant F protein according to any one of claims 1 to 6, characterized in that The recombinant F protein also includes a signal peptide; Preferably, the signal peptide is located at the N-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO: 11-23.

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

9. The polynucleotide according to claim 8, characterized in that The polynucleotide is a DNA molecule or an mRNA molecule; Preferably, the DNA molecule comprises a DNA sequence as shown in one of SEQ ID NOs: 105-180, or consists of the same; Preferably, the mRNA molecule comprises or consists of an RNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 105-180.

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

12. A host cell transformed or transfected with the polynucleotide according to claim 8 or 9, the nucleic acid construct according to claim 10 or the expression vector according to 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 bacterial cell is an Escherichia coli cell.

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

14. The use of the RSV-B recombinant F protein as described in any one of claims 1 to 7, the polynucleotide as described in claim 8 or 9, the nucleic acid construct as described in claim 10, the expression vector as described in claim 11, the host cell as described in claim 12 or the RSV-B recombinant F protein trimer as described in 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 as described in any one of claims 1 to 7, a polynucleotide as described in claim 8 or 9, a nucleic acid construct as described in claim 10, an expression vector as described in claim 11, a host cell as described in claim 12 or the RSV-B recombinant F protein trimer as described in claim 13, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

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

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

18. The vaccine or immunogenic composition according to claim 15, which is a respiratory syncytial virus mRNA vaccine, comprising: (I) an mRNA sequence encoding the RSV-B recombinant F protein as described in any one of claims 1 to 7, preferably an mRNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 105 to 180; and (II) Lipid nanoparticles.

19. The vaccine or immunogenic composition according to claim 15, which is a respiratory syncytial virus viral vector vaccine, comprising: (1) Viral backbone vector; and (2) a DNA sequence encoding the RSV-B recombinant F protein according to any one of claims 1 to 7, which is constructed into the viral backbone vector, preferably a DNA sequence as shown in any one of SEQ ID NOs: 105 to 180; 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.

20. The vaccine or immunogenic composition according to any one of claims 15 to 19, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays and powder sprays; Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film-coated capsules, pellets, sublingual tablets and ointments; Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a pushable preparation.

21. The method for preparing the RSV-B recombinant F protein according to any one of claims 1 to 5, characterized in that: The preparation method comprises: Add a nucleotide sequence encoding a signal peptide to the 5' end of the codon-optimized nucleotide sequence encoding the RSV-B recombinant F protein as described in any one of claims 1 to 5, and add a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon to the 3' end, clone and express, screen the correct recombinant, and then transfect it into an expression system cell for expression, collect the cell culture supernatant, and isolate the RSV-B recombinant F protein therefrom; Optionally, the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; Further optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell; Further optionally, the bacterial cell is an Escherichia coli cell.

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