A respiratory syncytial virus recombinant fusion protein in a prefusion conformation, methods of making and uses thereof

Abstract

The present application relates to a kind of respiratory syncytial virus A subtype (RSV-A) recombinant F protein, polynucleotide for encoding it, nucleic acid construct comprising the polynucleotide, expression vector comprising the nucleic acid construct, the host cell transformed or transfected with above-mentioned polynucleotide, nucleic acid construct or expression vector, the stable trimer formed by it, immunogenic composition comprising any of the foregoing, and its purposes in the preparation for preventing and / or treating respiratory syncytial virus infection vaccine.The RSV-A recombinant F protein of the present application comprises at least one specific epitope of pre-fusion F protein, can form stable, pre-fusion conformation F protein trimer, and its expression is stable, form uniform, and yield is greatly improved;The F protein trimer formed has good immunogenicity, can stimulate the body to produce high level of antibody titer, and has great significance for the clinical treatment and prevention and control of 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 202211199073.9 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 recombinant F protein of respiratory syncytial virus subtype A (RSV-A), 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 in the increase in infant mortality. According to WHO estimates, 64 million children are infected with RSV worldwide each year, of which 160,000 children die from RSV infection. In 2020, the number of severe RSV infections in children under 5 years old 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 revealed that the annual incidence of RSV infection ranges from 2% to 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 a single serotype, with two major antigenic subgroups, A and B. Strains of both subtypes often co-circulate, but during epidemics, only one subtype typically dominates. In temperate regions, including my country, RSV infection exhibits a distinct seasonal pattern, with onset occurring in late autumn and early winter, peaking between mid-December and early February, and declining in late spring. Safe, effective, and affordable preventive treatments for RSV infection are urgently needed worldwide.

[0006] RSV, belonging to the genus Pneumovirus in the family Paramyxoviridae, is a polymorphic enveloped virus approximately 120–300 nm in diameter. It possesses a nonsegmented, negative-sense, single-stranded RNA (15–16 kb) encoding 11 proteins. The viral envelope comprises three proteins: the attachment (G) protein, the fusion (F) glycoprotein, and the small hydrophobic (SH) protein. The G protein plays a role in host cell attachment, while the F protein is responsible for fusion and cell entry; neither process requires the SH protein. Two RSV surface glycoproteins are the primary neutralizing antigens: the G protein exhibits high sequence diversity and determines the viral antigenic subtype (A and B); the fusion (F) protein is highly conserved between the two subtypes and is recognized by a broad range of cross-neutralizing antibodies. Due to its critical role in RSV entry and its highly conserved protein sequence, the RSV F protein is a target for neutralizing antibodies and a primary antigen for vaccine development. The F protein is a type I transmembrane protein synthesized as a 574-amino acid precursor protein, F0, which has five to six post-translational N-linked glycosylation modifications. To become a functional fusion protein, F0 is processed by cellular furin-like proteases at two polybasic sites in the trans-Golgi apparatus, generating F1 (amino acids 137-574), F2 (amino acids 26-109), and the Pep27 polypeptide. Ultimately, two disulfide-linked fragments, F1 and F2, are formed, each 55 kDa and 15 kDa in size, respectively. The homologous F protein trimer consists of two conformations: prefusion and postfusion. The prefusion F protein is a metastable structure. Upon fusion of the virus and the cell, it transforms into a stable postfusion F protein, a process that can also occur spontaneously. Because the epitopes of highly active neutralizing antibodies against the F protein are primarily located on the prefusion F protein, maintaining the F protein in the prefusion conformation is crucial for RSV vaccine development. To date, no RSV vaccine is commercially available. 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 A (RSV-A) (hereinafter referred to as "RSV-A-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 A (RSV-A), which is a recombinant F protein based on respiratory syncytial virus subtype A, also referred to herein as RSV-A recombinant protein or RSV-A recombinant F protein, the RSV-A recombinant F protein comprising an RSV-A-F1 peptide segment and an RSV-A-F2 peptide segment, wherein the RSV-A-F1 peptide segment and / or RSV-A-F2 peptide segment comprise at least one mutation to proline relative to the corresponding peptide segment of the wild-type RSV-A F protein.

[0010] In a specific embodiment, the RSV-A-F1 peptide segment corresponds to the amino acid fragment at positions 26-99, 26-103, 26-108 or 26-97 in the amino acid sequence of the wild-type RSV-AF protein as shown in SEQ ID NO: 1, and the RSV-A-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-A F protein as shown in SEQ ID NO: 1, and the RSV-A-F1 peptide segment and / or RSV-A-F2 peptide segment contain one or more mutations to proline relative to the corresponding peptide segment of the wild-type RSV-A 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-AF protein as shown in SEQ ID NO: 1:

[0012] K65P, N67P, 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] N67P; for example, in the RSV-A recombinant F protein Fm 1 as shown in SEQ ID NO: 29;

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

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

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

[0018] N67P+L141P; for example, in the RSV-A recombinant F protein Fm 5 shown in SEQ ID NO: 33;

[0019] N67P+Q279P; for example, in the RSV-A recombinant F protein Fm 6 as shown in SEQ ID NO:34;

[0020] N67P+S377P; for example, in the RSV-A recombinant F protein Fm 7 as shown in SEQ ID NO:35;

[0021] L141P+Q279P; for example, in the RSV-A recombinant F protein Fm 8 as shown in SEQ ID NO:36;

[0022] L141P+S377P; for example, in the RSV-A recombinant F protein Fm 9 as shown in SEQ ID NO: 37;

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

[0024] N67P+L141P+Q279P; for example, in the RSV-A recombinant F protein Fm 11 as shown in SEQ ID NO: 39;

[0025] N67P+L141P+S377P; for example, in the RSV-A recombinant F protein Fm 12 as shown in SEQ ID NO:40;

[0026] N67P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm 13 as shown in SEQ ID NO:41;

[0027] L141P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm 14 shown in SEQ ID NO:42;

[0028] N67P+L138P+G139P; for example, in the RSV-A recombinant F protein Fm 15 as shown in SEQ ID NO:43;

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

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

[0031] N67P+L138P+G139P+Q279P; for example, in the RSV-A recombinant F protein Fm18 shown in SEQ ID NO:46;

[0032] N67P+L138P+G139P+S377P; for example, in the RSV-A recombinant F protein Fm19 shown in SEQ ID NO:47;

[0033] N67P+L141P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm20 shown in SEQ ID NO:48;

[0034] L138P+G139P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm21 shown in SEQ ID NO:49;

[0035] N67P+L138P+G139P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm 22 as shown in SEQ ID NO:50;

[0036] N67P+L138P+G139P+L141P+Q279P+S377P; for example, in the RSV-A recombinant F protein Fm 23 as shown in SEQ ID NO:51;

[0037] Optionally, the N67P mutation in the above mutation or combination of mutations may be replaced by a K65P mutation or a D73P mutation;

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

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

[0040] For example, in some embodiments, the above mutations further introduce an S215P mutation, for example, in RSV-A 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 above mutations further introduce an E161P mutation, for example, in RSV-A recombinant F protein Fm 35-46 with an amino acid sequence as shown in any one of SEQ ID NOs: 63-74; in other embodiments, the above mutations further introduce an I214P mutation, for example, in RSV-A recombinant F protein Fm 47 with an amino acid sequence as shown in SEQ ID NO: 75; in other embodiments, the above mutations further introduce an N216P mutation, for example, in RSV-A recombinant F protein Fm 48 with an amino acid sequence as shown in SEQ ID NO: 76; in other embodiments, the above mutations further introduce a Q210P mutation, for example, in RSV-A recombinant F protein Fm 49-51 with an amino acid sequence as shown in any one of SEQ ID NOs: 77-79.

[0041] In some embodiments, the RSV-A-F1 peptide segment and the RSV-A-F2 peptide segment are directly linked, for example, in RSV-A recombinant F protein Fm 70-72.

[0042] In other embodiments, the RSV-A-F1 peptide segment and the RSV-A-F2 peptide segment are connected by 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, GS (as shown in SEQ ID NO:8, in Fm 65-67 of RSV-A recombinant F protein), or GSGSGRS (as shown in SEQ ID NO:7, in Fm 68-69 of RSV-A recombinant F protein);

[0045] (ii) (GGGGS)n linker, wherein n=1-5, preferably 1-3; optionally, comprising one or more mutations to proline; in a specific embodiment, this type of linker is GGGGSGGGGSGGGGS (i.e., SEQ ID NO: 2, for example, in RSV-A recombinant F protein Fm 52-55 (such as SEQ ID NO: 80-83)), GGPGSGGGGSGGGGS (i.e., SEQ ID NO: 3, for example, in RSV-A recombinant F protein Fm 1-51 (such as SEQ ID NO: 29-79)), GGGGSPGGGSGGGGS (i.e., SEQ ID NO: 4, for example, in RSV-A recombinant F protein Fm 56 (such as SEQ ID NO: 84)), GGGGPGGGSGGGGS (i.e., SEQ ID NO: 5, for example, in RSV-A recombinant F protein Fm 57 (such as SEQ ID NO: 85)). NO:85)) or GGGGSGGGPSGGGGS (SEQ ID NO:6, for example in RSV-A recombinant F protein Fm 58 (such as SEQ ID NO:86));

[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: 9 (e.g., in RSV-A recombinant F protein Fm 59-61 (e.g., SEQ ID NOs: 87-89));

[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: 10 (for example, in RSV-A recombinant F protein Fm 62-64 (such as SEQ ID NO: 90-92)).

[0048] Preferably, the connecting bridge comprises or consists of an amino acid sequence selected from the group consisting of: a sequence as shown in SEQ ID NO: 2-10.

[0049] In a preferred embodiment, the RSV-A recombinant F protein comprises, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-100, 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: 29-100, and having the same or substantially the same immunogenicity.

[0050] Furthermore, in some embodiments, the RSV-A 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 NOs: 24-27; for example, in RSV-A recombinant F proteins Fm73-76 (as shown in SEQ ID NOs: 101-104), the trimer tags are SEQ ID NOs: 24-27, respectively;

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

[0053] Furthermore, in some embodiments, the RSV-A 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; for example, in RSV-A recombinant F protein Fm 73, the signal peptide is SEQ ID NO: 14; in RSV-A recombinant F protein Fm74-76, the signal peptide is SEQ ID NO: 11.

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

[0056] In a specific embodiment, the polynucleotide is a nucleotide sequence that has been 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 or consists of a DNA sequence as shown in one of SEQ ID NOs: 105-180.

[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 according to the second aspect, the nucleic acid construct according to the third aspect, or the expression vector according to 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 cells are Escherichia coli cells.

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

[0066] In the seventh aspect, the present invention provides the use of the RSV-A 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-A recombinant F protein trimer as described in the sixth aspect above in the preparation of a vaccine for preventing and / or treating novel coronavirus infection.

[0067] In an eighth aspect, the present invention provides a vaccine or immunogenic composition, comprising the RSV-A 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-A 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-A recombinant F protein as described in the first aspect above or the RSV-A 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-like series adjuvant.

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

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

[0072] (2) A DNA sequence encoding the RSV-A recombinant F protein as described in the first aspect constructed and incorporated 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-A 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 comprising:

[0078] (1) viral backbone vectors; and

[0079] (2) A DNA sequence encoding the RSV-A 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, and adeno-associated virus vector.

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

[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, a liquid for external use, an injectable or a pushable preparation.

[0085] In a ninth aspect, the present invention provides a method for preparing the RSV-A 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-A 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. Cloning and expression are performed, and the correct recombinant is screened. It is then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate and obtain the RSV-A recombinant F protein.

[0087] In one possible implementation of the above method, the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells; optionally, the bacterial cells are Escherichia coli cells.

[0088] Beneficial effects

[0089] When the natural RSV-A virus F protein antigen is recombinantly expressed in vitro, the pre-fusion conformation is unstable and the F antigen protein cannot be obtained. In the present invention, by introducing one or more mutations to proline in the F1 and / or F2 peptide segments of the respiratory syncytial virus subtype A F protein, a stable, pre-fusion conformation of the RSV-A recombinant F protein of the present invention is formed; experiments have shown that the formed RSV-A recombinant F protein contains at least one specific epitope of the pre-fusion F protein, and its expression is stable, uniform in form, and the yield is greatly improved; in addition, the RSV-A recombinant F protein of the present invention has good immunogenicity and can stimulate the body to produce high levels of antibody titers (after boosting immunization with the recombinant protein without adding an 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 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 illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

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

[0092] Figure 2 In Example 3, the expression of RSV-A 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-A recombinant F protein) and the vertical axis represents the OD450nm absorbance value.

[0093] Figure 3 In Example 3, the expression of a portion of the RSV-A recombinant F protein in the diluted supernatant was detected by ELISA using the D25 monoclonal antibody. The horizontal axis represents the Log10 value of the dilution factor of the cell supernatant, the vertical axis represents the OD450 nm absorbance, and the legend on the right indicates the experimental group (i.e., the detected RSV-A recombinant F protein).

[0094] Figure 4 In Example 3, the expression of a portion of the RSV-A recombinant F protein in the diluted supernatant was detected by ELISA using palivizumab, wherein the abscissa is the Log10 value of the dilution multiple of the cell supernatant, the ordinate is the OD450nm absorbance value, and the legend on the right shows the experimental group (i.e., the RSV-A recombinant F protein detected).

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

[0096] Figure 6 In Example 6, the titer level of specific antibody IgG in the serum of mice after primary immunization and booster immunization of mice using RSV-A recombinant F protein as an immunogen as described in Example 5 was detected by ELISA experiment, wherein the horizontal axis is the grouping in Table 1 and the vertical axis is the antibody titer.

[0097] Figure 7 This is a structural diagram of the RSV-A recombinant F protein Fm 30 analyzed in Example 7.

[0098] Figure 8In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 1-10 and WT recombinant F proteins in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. 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 RSV-A recombinant F protein detected).

[0099] Figure 9 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 11-20 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. The abscissa is the Log10 value of the dilution multiple of the cell supernatant, and the ordinate is the OD450 nm absorbance value. The legend on the right shows the experimental group (i.e., the RSV-A recombinant F protein detected).

[0100] Figure 10 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 21-30 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. 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-A recombinant F protein detected).

[0101] Figure 11 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 31-40 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. 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-A recombinant F protein detected).

[0102] Figure 12In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 41-50 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. The horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, and the vertical axis is the OD450 nm absorbance value. The legend on the right shows the experimental group (i.e., the RSV-A recombinant F protein detected).

[0103] Figure 13 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 51-60 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. The horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, and the vertical axis is the OD450 nm absorbance value. The legend on the right shows the experimental group (i.e., the RSV-A recombinant F protein detected).

[0104] Figure 14 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 61-70 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. 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-A recombinant F protein detected).

[0105] Figure 15 In Example 9, D25, AM22, AM14, palivizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-A-Fm 71-76 recombinant F protein in the diluted supernatant and the binding of each recombinant F protein to the above antibodies by ELISA. The horizontal axis is the Log10 value of the dilution multiple of the cell supernatant, and the vertical axis is the OD450 nm absorbance value. The legend on the right shows the experimental group (i.e., the RSV-A recombinant F protein detected).

[0106] Figure 16 The SDS-PAGE detection results of the isolated and purified RSV-A-Fm29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins in Example 10 are shown.

[0107] Figure 17 The Western Blot detection results of the isolated and purified RSV-A-Fm29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins in Example 10 are shown.

[0108] Figure 18 In Example 11, AM22 monoclonal antibody was used to detect the binding of RSV-A-Fm29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins to the antibody before and after storage at 30°C for 4 weeks. The horizontal axis represents the protein concentration and the vertical axis represents the absorbance at OD450 nm.

[0109] Figure 19 In Example 11, palivizumab was used to detect the binding of RSV-A-Fm29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins to the antibody before and after storage at 30°C for 4 weeks, where the abscissa is the protein concentration and the ordinate is the OD450 nm absorbance.

[0110] Figure 20 In Example 13, the titer levels of specific binding antibodies IgG in the serum of mice after primary immunization and booster immunization of mice using RSV-A-Fm29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins as immunogens, respectively, as described in Example 12, were detected by ELISA experiments, wherein the horizontal axis represents the vaccine grouping and the vertical axis represents the antibody titer.

[0111] Figure 21 In Example 14, the neutralizing antibody levels against respiratory syncytial virus in the serum of mice after the mice were boosted with RSV-A-Fm29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant F proteins as immunogens described in Example 12 were detected by a microneutralization experiment, wherein the horizontal axis represents the vaccine grouping and the vertical axis represents the neutralizing antibody titer.

[0112] Figure 22In Example 15, the viral load of the lung tissue of mice immunized with the vaccine described in Example 12 and challenged with respiratory syncytial virus by intranasal drops was detected by plaque assay, wherein the horizontal axis represents the vaccine group and the vertical axis represents the viral load.

[0113] Figure 23 This is a diagram of the structure of the recombinant F protein RSV-AFm47 analyzed in Example 16. DETAILED DESCRIPTION

[0114] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0115] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0116] Unless expressly 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 other components.

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

[0118] In this example, 76 RSV-A recombinant F proteins of the present invention were designed and constructed, which were respectively named RSV-A-Fm 1-76, and their amino acid sequences were shown in SEQ ID NOs: 29-104, respectively; wherein, a signal peptide sequence as shown in SEQ ID NO: 11 was added to the N-terminus of the amino acid sequence of RSV-A-Fm 1-72 (as shown in SEQ ID NOs: 29-100), and a trimer tag as shown in SEQ ID NO: 24 and a histidine tag as shown in SEQ ID NO: 28 were added to the C-terminus; and RSV-A-Fm73-76 (as shown in SEQ ID NOs: 101-104) itself includes a signal peptide at the N-terminus and a trimer tag sequence and a histidine tag sequence at the C-terminus.

[0119] According to the codon preference of mammalian cells, the nucleic acid sequence encoding the RSV-A recombinant F protein of the present invention, RSV-A-Fm 1-76, was optimized to obtain optimized encoding nucleic acid sequences, as shown in SEQ ID NOs: 105-180, respectively. An EcoRI restriction site sequence, a Kozak sequence, and a nucleic acid coding sequence for a signal peptide were added to the 5' end of the nucleic acid sequences encoding SEQ ID NOs: 105-176, and a trimer tag nucleic acid coding sequence, a His tag nucleic acid coding sequence, a stop codon, and an XhoI restriction site sequence were added to the 3' end. The resulting sequences were then commissioned to be synthesized by GenScript Biotech Co., Ltd., and then ligated to the pCAGGS vector through the EcoRI and XhoI restriction sites to obtain expression plasmids expressing the recombinant protein RSV-A-Fm1-72. The sequences of SEQ ID NOs: 105-176 were added to the 5' end of the nucleic acid sequences encoding SEQ ID NOs: 105-176, and a trimer tag nucleic acid coding sequence, a His tag nucleic acid coding sequence, a stop codon, and an XhoI restriction site sequence were added to the 3' end of the nucleic acid sequences encoding SEQ ID NOs: 105-176. After adding the EcoRI restriction site sequence and Kozak sequence to the 5' end of the nucleic acid sequence encoding NO:177-180, and adding the stop codon and XhoI restriction site sequence to its 3' end, we commissioned GenScript Biotech Co., Ltd. to perform gene synthesis. Then, we ligated it to the pCAGGS vector through the EcoRI and XhoI restriction sites to obtain an expression plasmid expressing the recombinant protein RSV-A-Fm 73-76.

[0120] Example 2: Expression and purification of detection antibodies

[0121] 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.

[0122] Antibody expression

[0123] 14-16 hours before transfection, split the 293T cells with higher density into plates (for example, a 10cm culture dish that is 100% confluent with 293T cells is passaged at a ratio of 1:3). 14-16 hours later, when the cell density reaches over 70%, transfection can be performed. During transfection, the heavy chain and light chain plasmids of the antibody are co-transfected into 293T cells at a ratio of 2:3. 4-6 hours after transfection, the cells are washed twice with PBS and replaced with serum-free DMEM medium for continued culture. The cell supernatants are collected on days 3 and 7 after transfection, centrifuged to remove cell debris, and the antibody supernatants obtained from the two washes are combined for subsequent antibody protein purification.

[0124] Antibody purification

[0125] A Protein A (5 ml) HP affinity column (GE) was connected to an AKTA Purifier / Explorer / FPLC / START (GE). The instrument was operated as follows: 20% ethanol was flushed out of the column with water, and the column was equilibrated with 20 mM Na3PO4, pH 7.0 buffer. After the conductivity on the instrument stabilized, the antibody supernatant was injected via a 10 ml loop loading method to bind to Protein A at a flow rate of 2 ml / min. After UV stabilization, approximately 0.8 ml of 1 M Tris pH 9.0 buffer was added to the subsequent collection tube (collection volume approximately 3.2 ml). The program was then changed to 100% 0.1 M Gly, pH 3.0, to elute the antibody bound to the column. The eluate was collected and then replaced with PBS by concentration and exchange. The resulting antibody solution could be used directly or stored in aliquots at -80°C for later use.

[0126] Example 3: Expression and conformation identification of RSV-A-Fm recombinant protein

[0127] In this example, HEK293T cells were transfected with expression plasmids of some RSV-A-Fm recombinant proteins (Fm 11, 12, 14, 19, 20, 22-58, 60, 63, 65-67, 69, 72-76) constructed in Example 1 to express RSV-A-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.

[0128] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS, and before transfection, the cell density was increased to 70% or more; HEK293T cells were transfected with the RSV-A-Fm recombinant protein Fm 11, 12, 14, 19, 20, 22-58, 60, 63, 65-67, 69, and 72-76 expression plasmids constructed in Example 1. 4-6 hours after transfection, the cell culture medium was replaced with serum-free DMEM, and culture was continued for 3 days. The cell culture supernatant was collected and the antigen protein expression was detected by ELISA using D25 monoclonal antibody and palivizumab, respectively. The specific detection method is as follows:

[0129] (1) The purified detection antibodies (D25 monoclonal antibody and palivizumab) obtained in Example 2 were diluted to 1 μg / ml using ELISA coating solution (Solebol, C1050). 100 μl of the solution was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0130] (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, 200 μl per well, block, and leave at room temperature for 1 hour; after blocking, wash once with PBS solution;

[0131] (3) During the blocking period of step (2), the cell culture supernatant was diluted with blocking solution, starting from 5-fold and then diluted in a 3-fold gradient; then, 100 μl of the culture supernatant of each RSV-A recombinant F protein to be tested or the culture supernatant of each dilution of a portion of RSV-A recombinant F protein (RSV-A-Fm 28, 30, 32, 33, 34, 53) was added to each well of the ELISA plate. For the negative control, blocking solution was added, and the cells were incubated at 37°C for 2 hours, followed by washing 4 times with PBST;

[0132] (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 2M hydrochloric acid to terminate the reaction, and measure the OD450 value on a microplate reader.

[0133] The expression results of each RSV-A recombinant F protein in the supernatant stock solution are as follows Figure 1 and 2 As shown, from Figure 1 and 2 The results show that compared with NC and wild-type RSV-AF proteins, the expression of all RSV-A-Fm recombinant proteins tested was greatly improved; moreover, all RSV-A-Fm recombinant proteins tested can bind to both palivizumab (which recognizes both the pre-fusion conformation of F protein and the post-fusion conformation of F protein) and D25 monoclonal antibody (which only recognizes the pre-fusion conformation of F protein), which indicates that the RSV-A-Fm recombinant protein of the present invention is in the pre-fusion conformation.

[0134] The expression results of RSV-A recombinant F protein RSV-A-Fm 28, 30, 32, 33, 34, and 53 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-A-Fm 28, 30, 32, 33, 34, and 53 cells was diluted 1000 times, both D25 monoclonal antibody and palivizumab could still detect the protein, indicating that RSV-A-Fm28, 30, 32, 33, 34, and 53 had a high expression level.

[0135] Example 4: Expression, purification and molecular weight identification of RSV-A-Fm recombinant protein

[0136] In this example, the RSV-A-Fm 30, 33, 34, and 53 recombinant protein expression plasmids constructed in Example 1 were used to transfect HEK293T cells to express RSV-A-Fm 30, 33, 34, and 53 recombinant proteins, which were then purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The target protein was collected and subjected to analytical ultracentrifugation to determine the molecular weight of the expressed recombinant protein.

[0137] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS to a cell density of over 70% before transfection. HEK293T cells were transfected with the RSV-A-Fm 30, 33, 34, and 53 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 cells were cultured for a further 3 days. The supernatant was collected, and DMEM medium was added again. The cells were cultured for a further 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 supernatant was filtered through a 0.22 μm filter to allow the target protein to bind to a HisTrap Excel column (5 mL, GE Healthcare). Non-specifically bound proteins were then eluted with an eluent containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 30 mM imidazole, and the target protein was eluted again 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 subjected to molecular sieve chromatography (Superdex 200 Increase 10 / 300 GL or Superdex 200 Hiload 16 / 60, GE Healthcare) to obtain purified RSV-A-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.

[0138] The UV absorption diagram of molecular sieve chromatography is as follows Figure 5 As shown in the left picture, Figure 5 The left figure shows that the eluates of the four recombinant proteins RSV-A-Fm 30, 33, 34, and 53 are all single peaks with no or almost no impurity peaks, indicating that these recombinant proteins do not form or only form very small amounts of aggregates; a single target protein peak was collected for analytical ultracentrifugation, and the results are as follows: Figure 5 As shown in the figure on the right, Figure 5 The right figure shows that the measured molecular weights of RSV-A-Fm30, 33, 34, and 53 are 168kDa, 173kDa, 181kDa, and 175kDa, respectively, which are consistent with the theoretical molecular weight of the trimer, indicating that the above-mentioned RSV-A-Fm 30, 33, 34, and 53 proteins are all trimers.

[0139] Example 5: RSV-A-Fm recombinant protein immunization mouse experiment

[0140] In this example, RSV-A-Fm 30, 33, 34, and 53 recombinant proteins obtained in Example 4 were used to immunize mice, respectively. The experimental mice were BALB / c mice aged 4-6 weeks with an average weight of 15-20 g.

[0141] Specifically, the RSV-A-Fm 30, 33, 34, and 53 recombinant proteins obtained in Example 4 were used to immunize mice. Each recombinant protein was further divided into a pure protein immunization group and an adjuvant-added group. The adjuvant was an MF59-like adjuvant, AddaVax. The immunization grouping, the immunogen used in each group, the immunogen dose used in each group, and the adjuvant are shown in Table 1. Blank cells indicate "none." Each RSV-A-Fm recombinant protein was diluted to the desired concentration in physiological saline. The adjuvant-added group was further emulsified with the adjuvant. Six BALB / c mice (average weight 15-20 g) aged 4-6 weeks were included in each group.

[0142] Table 1

[0143]

[0144] Each group of mice was injected intramuscularly with recombinant protein vaccine or normal saline on days 0 and 14, respectively, with a volume of 100 μl for each immunization. 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.

[0145] Example 6: ELISA test to detect vaccine-induced specific antibody titers

[0146] In this example, the titer of specific IgG antibodies in the sera of mice immunized with RSV-A-Fm 30, 33, 34, and 53 recombinant proteins in Example 5 was detected by ELISA experiments.

[0147] Specifically, the following procedures were adopted:

[0148] (1) RSV-A-Fm 30, 33, 34, and 53 recombinant proteins prepared in Example 4 were diluted to 3 μg / ml using ELISA coating solution (Solarbio, C1050). 100 μl of the solution was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0149] (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 to block; after blocking, wash once with PBS solution;

[0150] (3) During the blocking period of step (2), the mouse serum samples were diluted with blocking solution, starting from 10 times and then diluted in a 2-fold gradient; then, 100 μl of immune serum dilution solution 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;

[0151] (4) Add HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 in 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 a microplate reader.

[0152] 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.

[0153] The results are as follows Figure 6 As shown, Figure 6 The results showed that the four recombinant proteins RSV-A-Fm 30, 33, 34, and 53 all had good immunogenicity. Among them, after boosting immunization with the recombinant proteins without adding adjuvants, the antigen-specific antibody titers induced by the four proteins all reached more than 10,000. After adding AddaVax adjuvant, the antigen-specific antibody titers could be further increased by 10-100 times.

[0154] Example 7: RSV-A-Fm 30 crystal structure analysis

[0155] RSV-A-Fm 30 protein was expressed and purified according to the method described in Example 4. After purification, the target protein peak was collected, the protein was concentrated to 10 mg / ml, and the protein was mixed with the crystallization pool solution at a volume ratio of 1:1, and then Protein crystal screening was performed using a protein crystallization screening liquid workstation (TTP LabTech). Crystals were grown at 18°C ​​and diffraction-capable crystals were obtained. Crystals were collected at the Shanghai Synchrotron Radiation Facility (SSRF) and approximately The data were analyzed by HKL2000 software, and the structure of RSV-A-Fm30 was finally solved by molecular replacement method using the structure of DS-Cav2 as a template (PDB: 5K6I).

[0156] The resolved structure of RSV-A-Fm 30 is shown in Figure 7 As shown, from Figure 7 The results showed that RSV-A-Fm 30 is an F protein in the prefusion conformation.

[0157] Example 8: Expression and purification of detection antibodies

[0158] 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 (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 the detection antibodies for RSV F protein, AM22, AM14, MPE8, 101F and hRSV90 monoclonal antibodies, were constructed respectively.

[0159] The specific methods for antibody expression, isolation and purification are as described in Example 2. The obtained antibody solution can be used directly or stored in a -80°C refrigerator for later use.

[0160] Example 9: Expression and conformation identification of RSV-A-Fm recombinant protein

[0161] In this example, all RSV-A-Fm recombinant protein (Fm 1-76) expression plasmids constructed in Example 1 were used to transfect HEK293T cells to express RSV-A-Fm recombinant protein. ELISA was performed using the detection antibodies prepared in Examples 2 and 8 to determine the expression level and conformation of the expressed recombinant protein. For specific methods, see Example 3.

[0162] The expression results of each RSV-A recombinant F protein in the supernatant stock solution are as follows Figure 8-15 As shown by Figure 8-15It can be seen that compared with the wild-type RSV-AF protein, except for the slightly increased expression levels of RSV-A-Fm2, Fm25, and Fm75 recombinant proteins, the expression levels of all other recombinant F proteins were greatly increased; moreover, all the RSV-A-Fm recombinant proteins tested could bind to the corresponding monoclonal antibodies of the common epitope II before and after fusion (i.e., palivizumab) and the corresponding monoclonal antibodies of the common epitope IV (i.e., 101F monoclonal antibody), as well as the specific epitope of the prefusion conformation. The corresponding monoclonal antibodies (i.e., D25 and AM22 monoclonal antibodies), the corresponding monoclonal antibodies of the trimer-dependent and pre-fusion conformation-specific spatial conformation epitope (i.e., AM14 monoclonal antibody), the corresponding monoclonal antibodies of the pre-fusion conformation-specific epitope V (i.e., hRSV90 monoclonal antibody), and the corresponding monoclonal antibodies of the pre-fusion conformation-specific epitope III (i.e., MPE8 monoclonal antibody) indicate that the RSV-A-Fm recombinant protein of the present invention is a pre-fusion conformation.

[0163] Example 10: RSV-A-Fm recombinant protein expression, purification and SDS-PAGE identification

[0164] In this example, the RSV-A-Fm 29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant protein expression plasmids constructed in Example 1 were transfected into HEK293T cells to express RSV-A-Fm 29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant proteins, and purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The specific operating steps are described in Example 4. After gel filtration chromatography, each recombinant F protein eluted only a single peak, with no or only very small miscellaneous peaks, indicating that these recombinant F proteins did not form or formed only very small amounts of aggregates.

[0165] The target protein peak was collected and analyzed by SDS-PAGE. Figure 16 As shown, Figure 16 The results showed that RSV-A-Fm 29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant proteins all showed one obvious protein band, indicating that they all had high purity; in addition, the molecular weights of different recombinant F proteins were slightly different, among which the molecular weights of RSV-A-Fm 29, 32, 38, 39, 40, 41, 46, 47, 48, 50, 51, 55, 63, and 64 were between 55 and 72 KDa, and the molecular weights of RSV-A-Fm 60, 67, 69, 70, 71, and 72 were slightly smaller.

[0166] In addition, since all RSV-A-Fm recombinant F proteins are histidine-tagged recombinant proteins, in this example, an anti-histidine tag antibody coupled with horseradish peroxidase was used for Western Blot identification. Figure 17 As shown; Figure 17 It was shown that the expression of histidine tags was detected in each RSV-A-Fm recombinant F protein collection fluid, indicating that the expression of these RSV-A-Fm recombinant F proteins was consistent with expectations and was correctly expressed.

[0167] Example 11: Stability test of RSV-A-Fm recombinant protein

[0168] RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, 72 recombinant proteins obtained in Examples 4 and 10 were sterile filtered and stored at 30°C. After 4 weeks, the recombinant proteins were detected by ELISA using AM22 monoclonal antibody (antibody against epitope ) and palivizumab (directed at epitope II) were tested by ELISA to determine the binding of antigen and antibody and the stability of the protein during long-term storage. The specific test method is as follows:

[0169] (1) The purified antibodies (palivizumab and AM22 monoclonal antibody) obtained in Examples 2 and 8 were diluted to 1 μg / ml using ELISA coating solution (Solebol, C1050), and 100 μl was added to each well of a 96-well ELISA plate (Corning, 3590). The plate was incubated at 4°C for 12 hours.

[0170] (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, 200 μl per well, block, and leave at room temperature for 1 hour; after blocking, wash once with PBS solution;

[0171] (3) During the blocking period of step (2), the RSV-A-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-A-Fm recombinant protein was added to each well of the ELISA plate. The negative control was to add blocking solution, incubate at 37°C for 2 hours, and then wash 4 times with PBST;

[0172] (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 2M hydrochloric acid to terminate the reaction, and measure the OD450 value on a microplate reader.

[0173] The ELISA test results using AM22 monoclonal antibody and palivizumab were as follows: Figure 18 and 19 As shown, Figure 18 and 19 It was shown that compared with the initial protein at week 0, after storage at 30°C for 4 weeks, the binding of RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant proteins to AM22 antibody and palivizumab did not change significantly, indicating that these RSV-A-Fm recombinant proteins have good stability.

[0174] Example 12: RSV-A-Fm recombinant protein immunization mouse experiment

[0175] The RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 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 weight of 15-20 g.

[0176] Specifically, mice were immunized with the aforementioned RSV-A-Fm recombinant F protein at a dose of 12 μg per immunization. The vaccine was formulated as follows: each RSV-A-Fm recombinant protein was diluted to the desired concentration in saline. The aluminum hydroxide adjuvant was first mixed with the antigen protein, followed by the CpG adjuvant. A placebo group received saline. Four BALB / c mice (average weight 15-20 g) aged 6-8 weeks were used in each group.

[0177] Each group of mice was injected intramuscularly with recombinant protein vaccine or normal saline on days 0 and 14, respectively, with a volume of 100 μl for each immunization. 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.

[0178] Example 13: ELISA assay to detect vaccine-induced antigen-specific antibody titers

[0179] In this example, an ELISA assay was performed to measure the titer (Log antibody titer) of specific IgG antibodies in the serum of mice immunized with the RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant protein vaccines in Example 12. The specific method and the definition of the antibody titer values ​​are described in Example 6.

[0180] The results are as follows Figure 20 As shown, Figure 20 It was shown that the antigen-specific antibody titers induced by all RSV-A-Fm recombinant F protein vaccines reached more than 10,000 after the first immunization, and the antigen-specific antibody titers induced after the second immunization could be further increased to more than 1,000,000, indicating that all RSV-A-Fm recombinant F proteins have excellent immunogenicity.

[0181] Example 14: Microneutralization assay to detect vaccine-induced neutralizing antibody titers

[0182] In this example, a microneutralization experiment was used to detect the neutralizing antibody titer in the serum of mice boosted with the RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 69, 70, 71, and 72 recombinant protein vaccines in Example 12.

[0183] Specifically, Vero cells were laid out one day in advance using a 96-well plate and used after the cell confluence reached 80-90%. The serum of mice immunized with two injections of the vaccine was diluted using DMEM medium containing 1% FBS and 1‰ antibiotics. The initial dilution was 100 times, and 8 gradients of 2-fold dilution were continuously performed. An equal volume of respiratory syncytial virus (RSV) of 100 TCID50 was mixed with the diluted immune mouse serum, incubated at 37°C for 1 hour, the cell culture supernatant was discarded, and 100 μl of the serum-virus mixture was added. An equal amount of respiratory syncytial virus (RSV) virus solution was added to the positive control, and an equal volume of cell culture medium without virus was added to the negative control. After incubation at 37°C for 5 hours, 100 ul of 1% FBS DMEM (containing double antibodies) was added and cultured at 37°C for 4 days. The supernatant was discarded, and the cells were washed once with PBS. 100 μl / well of a methanol / ethanol mixture (volume ratio 1:1) was added and fixed at room temperature for 10 minutes. The fixative was discarded again and the cells were washed once with PBS. Blocked with 200 μl / well of 5% skim milk at 37°C for 30 minutes. The blocking solution was discarded, and 100 μl / well of Palivuzumab (10 μg / ml) diluted with 5% skim milk was added and incubated at 37°C for 1 hour. The cells were washed three times with PBST (PBS with 0.05% Tween20), and then horseradish peroxidase-labeled goat anti-human IgG secondary antibody (purchased from Biyuntian Biotechnology) was added and incubated at 37°C for 1 hour. The cells were washed three times with PBST (PBS with 0.05% Tween20), and 50 μl / well of TMB colorimetric solution was added and placed at room temperature. After 6 minutes of reaction, 50 μl of 2M sulfuric acid stop solution was added and the OD was read using a microplate reader. 450 The average value of the cell well readings was taken as the background value, and the background value was subtracted from all well readings. The neutralization titer of each sample well was 100% minus the sample well value divided by the toxic well value * 100. The neutralization titer NT of each serum sample was calculated by the four-parameter fitting method. 50 value.

[0184] The neutralization titer results of the immune sera of each vaccine are as follows Figure 21 As shown, Figure 21 The results showed that compared with the placebo group, the neutralizing antibody titers in the mouse sera of all recombinant F protein vaccine immunization groups were significantly increased. Among them, the neutralizing antibody titers induced by RSV-A-Fm 29, 30, 32, 33, 34, 38, 39, 40, 41, 46, 47, 48, 50, 51, 53, 55, 60, 63, 64, 67, 70, and 71 recombinant F protein vaccines were above 3000, indicating that all RSV-A-Fm recombinant F protein vaccines have good immunogenicity.

[0185] Example 15: Evaluation of vaccine immune protection effect

[0186] For each vaccine-immunized mouse in Example 12, the respiratory syncytial virus Long strain was challenged by nasal drops on the 4th week after the secondary immunization, and the challenge dose was 10^4 PFU / 50μl / mouse. On the 5th day after the challenge, the mice were euthanized and the lungs were taken. The lungs of four mice in each group were weighed and recorded, and then DMEM medium was added and homogenized using a tissue homogenizer. The lung tissue supernatant was obtained after centrifugation at 5000g / min for 10 minutes. The viral load of respiratory syncytial virus in the lung tissue supernatant sample was detected by plaque assay, and the viral copy number per gram of lung tissue was finally calculated.

[0187] The specific method of the plaque assay is as follows: BHK cells were plated in a 12-well plate one day in advance, with 1*10 cells per well. 5 For each well, a 10-fold dilution of the tissue stock solution was prepared: 50 μl of lung tissue supernatant was added to 450 μl of DMEM medium and mixed thoroughly. Six 10-fold dilutions were then made. The cells to be infected were washed twice with PBS. 400 μl of the diluted lung tissue supernatant was then used to infect the cells. The cells were incubated at 37°C for 2 hours. The cells were then washed with PBS and an equal volume of 2% sodium carboxymethylcellulose and 2X DMEM was added, with 1 ml added to each well. The cells were incubated at 37°C 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 5% skim milk in PBST, blocked at 37°C for 1 hour, 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 added with AEC substrate (purchased from BD Biosciences) for reaction. Brown spots were determined as positive spots, and the PFU value of each sample was counted.

[0188] The results are as follows Figure 22 As shown, Figure 22 It showed that after the placebo immunization group was challenged with the vaccine, high titers of respiratory syncytial virus were detected in the lung tissue, while no virus was detected in all vaccine immunization groups. This shows that each vaccine can protect the body from respiratory syncytial virus infection after immunization and has a good protective effect.

[0189] Example 16: RSV-A-Fm 47 crystal structure analysis

[0190] RSV-A-Fm 47 recombinant protein was concentrated to 10 mg / ml, and the protein was mixed with the crystallization pool solution at a volume ratio of 1:1. Protein crystal screening was performed using a protein crystallization screening liquid workstation (TTP LabTech). Crystals were grown at 18°C ​​and diffraction-capable crystals were obtained. Crystals were collected at the Shanghai Synchrotron Radiation Facility (SSRF) and approximately The data were analyzed by HKL2000 software, and the crystal structure of RSV-A-Fm 47 was finally solved by molecular replacement method using the structure of DS-Cav2 as a template (PDB: 5K6I).

[0191] The resolved structure of RSV-A-Fm 47 is shown in Figure 23 As shown, Figure 23 It was shown that the RSV-A-Fm 47 protein was the F protein in the prefusion conformation.

[0192] 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 A, characterized in that The amino acid sequence of the respiratory syncytial virus subtype A recombinant F protein is shown in SEQ ID NO: 58, 61, 62, 67, 68, 74, 76, 78, 79, 81 or 83.

2. A recombinant F protein of respiratory syncytial virus subtype A, characterized in that: The respiratory syncytial virus subtype A recombinant F protein consists of the respiratory syncytial virus subtype A recombinant F protein according to claim 1 and a trimer tag; The trimer tag is located at the C-terminus, and its amino acid sequence is shown in any one of SEQ ID NOs: 24-27.

3. A recombinant F protein of respiratory syncytial virus subtype A, characterized in that The respiratory syncytial virus subtype A recombinant F protein consists of the respiratory syncytial virus subtype A recombinant F protein according to claim 1 or 2 and a signal peptide; The signal peptide is located at the N-terminus, and its amino acid sequence is shown in any one of SEQ ID NOs: 11-23.

4. A polynucleotide encoding the respiratory syncytial virus subtype A recombinant F protein according to any one of claims 1 to 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, wherein The DNA sequence of the DNA molecule is shown in one of SEQ ID NOs: 134, 137, 138, 143, 144, 150, 152, 154, 155, 157, and 159; And / or, the RNA sequence of the mRNA molecule corresponds to a DNA sequence as shown in one of SEQ ID NOs: 134, 137, 138, 143, 144, 150, 152, 154, 155, 157, 159.

7. A nucleic acid construct comprising the polynucleotide according to any one of claims 4 to 6, and at least one expression control element operably linked to the polynucleotide. 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 to 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 cells are 293T cells, 293F cells, or CHO cells; The bacterial cells are Escherichia coli cells.

12. A respiratory syncytial virus subtype A recombinant F protein trimer, which is formed by polymerizing three respiratory syncytial virus subtype A recombinant F proteins according to any one of claims 1 to 3.

13. Use of the respiratory syncytial virus subtype A recombinant F protein according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 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 to 11, or the respiratory syncytial virus subtype A 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 subtype A recombinant F protein according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 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 to 11, or the respiratory syncytial virus subtype A 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 subtype A recombinant F protein according to any one of claims 1 to 3 or the respiratory syncytial virus subtype A recombinant F protein trimer according to claim 12 and an adjuvant.

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

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

18. The vaccine or immunogenic composition according to claim 17, characterized in that The DNA sequence is shown as one of SEQ ID NOs: 134, 137, 138, 143, 144, 150, 152, 154, 155, 157, and 159; 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, comprising: (1) an mRNA sequence encoding the respiratory syncytial virus subtype A recombinant F protein according to any one of claims 1 to 3; and (II) Lipid nanoparticles.

20. The vaccine or immunogenic composition according to claim 19, characterized in that The mRNA sequence corresponds to a DNA sequence as shown in one of SEQ ID NOs: 134, 137, 138, 143, 144, 150, 152, 154, 155, 157, 159.

21. The vaccine or immunogenic composition according to claim 14, which is a respiratory syncytial virus viral vector vaccine comprising: (1) Viral backbone vector; and (2) A DNA sequence encoding the respiratory syncytial virus subtype A recombinant F protein according to any one of claims 1 to 3, constructed and incorporated into the viral backbone vector.

22. The vaccine or immunogenic composition according to claim 21, characterized in that The DNA sequence is shown as one of SEQ ID NOs: 134, 137, 138, 143, 144, 150, 152, 154, 155, 157, and 159; 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 to 22, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation.

24. The vaccine or immunogenic composition according to claim 23, characterized in that The nasal spray is selected from aerosols, sprays and powder sprays; The oral preparation is selected from tablets, powders, pills, powders, granules, soft capsules, hard capsules, film coatings and ointments; The parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, an injectable or a pushable preparation.

25. The method for preparing the recombinant F protein of respiratory syncytial virus subtype A according to claim 1, characterized in that: The preparation method comprises: A nucleotide sequence encoding a signal peptide is added to the 5' end of the codon-optimized nucleotide sequence encoding the respiratory syncytial virus subtype A recombinant F protein as claimed in claim 1, and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon are added to the 3' end, and cloning and expression are performed, and the correct recombinant is screened, and then it is transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate and obtain the respiratory syncytial virus subtype A recombinant F protein.

26. The preparation method according to claim 25, characterized in that The expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells.

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

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