A method for preparing a respiratory syncytial virus antigen and uses thereof

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a protein for preventing respiratory syncytial virus infection and application thereof. The present application is based on the sequence of RSV F protein, and a new F antigen sequence is obtained by removing the I and IV epitope sequences specific to post-F and the III epitope sequence part common to pre-F and post-F. Then, the two new F antigen sequences are connected together to form a new F antigen dimer, so as to maintain the pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and having high neutralization activity. The dimer antigen of the present application has the advantages of stimulating the body to produce more neutralizing antibodies and high yield.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to proteins for preventing respiratory syncytial virus infection and their applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the most common pathogens causing lower respiratory tract infections in infants and young children worldwide. In immunocompromised and weakened elderly populations, RSV infection can lead to severe lower respiratory tract symptoms, increasing the risk of death. Therefore, developing a safe, effective, and inexpensive vaccine to prevent RSV infection has become a pressing global issue.

[0003] RSV belongs to the Paramyxoviridae family and the Pneumovirus genus. Its genome is a single-stranded negative-sense virus, 15.2 kb in length, encoding 11 viral proteins. RSV has two glycoproteins on its surface: G and F. These are important RSV antigens and the main targets of neutralizing antibodies. The G protein shares only about 50% homology among different RSV subtypes; while the F protein is highly conserved, with only about 10% difference between different RSV subtypes. Therefore, the F protein is the main antigen for the design and development of broad-spectrum RSV vaccines.

[0004] During the fusion of the RSV F protein with the host cell membrane, it changes from a pre-F conformation with higher potential energy and lower stability to a post-F conformation with lower potential energy and relative stability. During this conformational change, pre-F specific epitopes... The V epitope is blocked, preventing the body from producing antibodies with high neutralizing activity; therefore, the development of RSV vaccines based on pre-F has become a hot research topic in the prevention of RSV infection. Although there are currently no clinically available RSV vaccines, several pre-F protein-based vaccines have entered clinical trials globally. These vaccines are all modified based on the monomeric structure of the F protein [Krarup A., et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nature Communications, 2015, 6:8143; Crank MC., et al. A proof of concept for structure-based vaccine design targeting RSV in humans. Science, 2019, 365, 505-509.] to maintain the conformation of pre-F. However, according to literature reports, these protein vaccines all have problems such as low yield (below 6 mg / L) [Joyce MG., et al. Iterative structure-based improvement of afusion-glycoprotein vaccine against RSV. Nature Structural & Molecular biology, 2016, 23(9): 811-820.] and inability to maintain the pre-F conformation [Crank MC., et al. Science, 2019, 365, 505-509.]. Summary of the Invention

[0005] To address the problems existing in the prior art, the inventors of this invention completed the present invention by modifying the RSV F protein sequence. Specifically, the key design of this invention is as follows: First, based on the RSV F protein sequence, a new F antigen sequence is obtained by removing the post-F-specific I and IV epitope sequences and the III epitope portion shared by pre-F and post-F; Second, the two new F antigen sequences are tandemly linked to form a new F antigen dimer. These two design features enable the new F antigen dimer to maintain the pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and possessing high neutralizing activity.

[0006] This invention provides a method for preparing respiratory syncytial virus antigen, wherein the respiratory syncytial virus antigen is obtained by expressing the following antigen-encoding gene:

[0007] The genes of RSV F protein monomers with the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F removed, or the genes of RSV F protein monomers tandemly in two or more forms, are used to obtain respiratory syncytial virus antigen encoding genes.

[0008] Preferably, based on the F protein sequence of the RSV A2 strain (preferably its amino acid sequence as shown in SEQ ID NO: 1), the N104-V147 segment (specifically as shown in SEQ ID NO: 2) and the V308-S552 segment (specifically as shown in SEQ ID NO: 3) are removed; a linker sequence (e.g., the two amino acids GS) is inserted between T103 and G148 to obtain a gene containing the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F; or the obtained new RSV F protein monomers are tandemly linked in two or more sequences to obtain a respiratory syncytial virus antigen encoding gene.

[0009] Preferably, the amino acid sequence with the I and IV epitopes specific to post-F removed is F387-D392 (FNPKYD) and K427-S436 (KNRGIIKTFS), and the amino acid sequence with the III epitope shared by pre-F and post-F is V40-L45 (VSKGYL).

[0010] The amino acid sequence of the RSV F protein monomer, which has removed the I and IV epitopes specific to post-F and the III epitope common to pre-F and post-F, is shown in SEQ ID NO: 4.

[0011] More preferably, the amino acid sequence of the respiratory syncytial virus antigen obtained by tandemly connecting two monomers is shown in SEQ ID NO: 5.

[0012] More preferably, a tag sequence and a stop codon coding nucleotide sequence are added to the 3' end.

[0013] Specifically, the nucleotide sequence encoding the tag sequence is a nucleic acid sequence encoding 6 histidine residues, preferably as shown in SEQ ID NO: 6.

[0014] Furthermore, the encoding nucleotide sequence of the stop codon is a single stop codon.

[0015] In a specific embodiment, the respiratory syncytial virus antigen encoding gene is expressed in a host cell using a recombinant expression vector to obtain the respiratory syncytial virus antigen; specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as pCAGGS vector, and the host cell is a mammalian cell such as 293F cell; optionally, the method further includes the steps of collecting cell supernatant and purifying the respiratory syncytial virus antigen after expression.

[0016] The present invention provides a respiratory syncytial virus antigen obtained by the preparation method described above, preferably having an amino acid sequence as shown in SEQ ID NO: 5, or including a tag sequence as shown in SEQ ID NO: 6.

[0017] Furthermore, the encoding gene of the respiratory syncytial virus antigen is provided.

[0018] The present invention also provides a recombinant expression vector containing the aforementioned coding gene and a recombinant host cell. Specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as the pCAGGS vector, and the host cell is a mammalian cell such as 293F cell.

[0019] The present invention also provides an antigen composition consisting of the respiratory syncytial virus antigen as the active ingredient and an adjuvant, preferably an aluminum adjuvant and an AddVax adjuvant.

[0020] The present invention also provides the use of the respiratory syncytial virus antigen in the preparation of reagents for immunizing animals to obtain antibodies.

[0021] The dimer antigen provided by this invention not only maintains its pre-F conformation in vitro and in vivo, thereby stimulating the body to produce antibodies specific to the pre-F epitope and possessing high neutralizing activity, but also has the advantage of stimulating the body to produce more neutralizing antibodies. Furthermore, experiments have demonstrated that the F antigen dimer of this invention also has the advantage of high yield (exceeding 30 mg / L in experiments). Attached Figure Description

[0022] Figure 1 Superdex 200Increase 10 / 300 (GE) molecular sieve chromatography and electrophoresis of RSV new F antigen dimer.

[0023] Figure 2 Schematic diagram of the immunization strategy.

[0024] Figure 3 Results of RSVF-specific IgG antibody titers induced in cotton rats after immunization with RSVF new antigen dimer.

[0025] Figure 41. In vivo conformational analysis of the protein after immunization with the new F antigen dimer of RSV in cotton rats (using ELISA to infer whether the antigen is pre-F or post-F in vivo).

[0026] Figure 5 Results of RSVA2 virus neutralizing antibody titers induced after rats were immunized with RSV neoF antigen dimer.

[0027] Figure 6 Results of RSVB9320 virus neutralizing antibody titers induced after rats were immunized with RSV new F antigen dimer.

[0028] Figure 7 Results of live virus titers in various tissues after re-challenge with RSV new F antigen dimer in cotton rats.

[0029] Figure 8 Pathological sections of lung tissue from rats after being immunized with RSV new F antigen dimer and then challenged. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but this should not be construed as limiting the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] The sequence information is as follows:

[0032] SEQ ID NO: 1 (Full-length sequence of F protein of RSV A2 strain):

[0033] NITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGS.

[0034] SEQ ID NO: 2 (amino acid sequence of segment N104-V147 of F protein of RSV strain A2):

[0035] NNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV.

[0036] SEQ ID NO: 3 (amino acid sequence of segment V308-S552 of F protein of RSV strain A2):

[0037] VIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGS.

[0038] SEQ ID NO: 4 (Amino acid sequence of the protein monomer of the present invention):

[0039] NITEEFYQTTASAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVS VLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYG.

[0040] SEQ ID NO: 5 (Amino acid sequence of the antigen of this invention, excluding the tag sequence):

[0041] NITEEFYQTTASAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGNITEEFYQTTASAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYG.

[0042] SEQ ID NO: 6 (amino acid sequence of the antigen of the present invention, containing histidine tag):

[0043] NITEEFYQTTASAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSV LTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGNIT EEFYQTTASAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFK VLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGHHHHHHH.

[0044] Example 1: Expression and purification of RSV novel F antigen dimer

[0045] Based on the F protein sequence of the RSVA2 strain (as shown in SEQ ID NO: 1), the N104-V147 segment (as shown in SEQ ID NO: 2) and the V308-S552 segment (as shown in SEQ ID NO: 3) were removed. A linker sequence (two amino acids GS) was inserted between T103 and G148 to obtain the amino acid sequence of the RSV F protein monomer that does not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F (as shown in SEQ ID NO: 4). The amino acid sequences of the obtained new RSVF protein monomer were then tandemly linked in pairs (as shown in SEQ ID NO: 5), and six histidine tags were added to the 3' end (as shown in SEQ ID NO: 6). After codon optimization, the respiratory syncytial virus antigen encoding gene was obtained.

[0046] The obtained respiratory syncytial virus antigen-encoding gene sequence was then cloned into the pCAGGS vector to obtain the pCAGGS-new F antigen dimer plasmid. The plasmid was then transfected into a 293F cell expression system for expression, and the cell supernatant was collected and purified after expression.

[0047] Chromatography was performed using Superdex 200 Increase 10 / 300 (GE) molecular sieves, and the elution peak around 13.5 mL was collected (e.g., Figure 1 SDS-PAGE analysis was performed on the protein sample (shown in the image). The protein in the elution volume of approximately 13.5 ml was about 55 kDa under both non-reducing conditions (without DTT) and reducing conditions (with DTT), confirming that the protein obtained from this elution peak is a new F antigen dimer.

[0048] Example 2: Affinity analysis of RSV novel F antigen dimer antibody

[0049] Using the Biacore 8K biomolecular interaction analysis system, the new RSV F antigen dimer obtained in Example 1 was immobilized on an SM chip. Then, various antibody Fab fragments targeting different epitopes of the F protein were flowed onto the chip surface, and the affinity between the antigen and various antibodies was measured.In this example, epitope I 4D7 [Flynn JA., et al. Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein. PLoS ONE, 2016, 11(10):e0164789.], epitope II Palivazumab [The IMPACT-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospi-talization from respiratory syncytial virus infection in high-risk infants. Pediatrics, 1998, 102, 531-537], epitope III MPE8 [Gilman MS. et al. Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein. PLoS Pathogen, 2015, 11:e1005035.], epitope IV 101F [McLellan JS. et al. Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. Journal of Virology, 2010, 84:12236-12244.], epitope V CR9501 [Gilman MS. et al. Transient opening of trimeric prefusion RSV F proteins. Nature Communications, 2019, 10:2105.] are selected in this example. Epitope Am22 [McLellan JS. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Scinece, 2013, 340(6136):1113-1117.]. Biacore 8K analysis was used to obtain the binding and affinity of the new RSV F antigen dimer to the antibody (as shown in Table 1). Blank cells indicate that the antigen and antibody do not bind.

[0050] Table 1. Binding and affinity of RSV novel F antigen dimer to antibodies.

[0051]

[0052] Example 3: RSV new F antigen dimer immunization experiment in cotton rats

[0053] MF59 (AddaVax used below is an MF59-like adjuvant) and aluminum adjuvant are two commonly used adjuvants approved by the SFDA. This invention selects these two adjuvants as vaccine adjuvants for animal experiments, which will have direct guiding significance for subsequent clinical trials. Therefore, this invention dilutes the antigen obtained in Example 1 with PBS solution to the required concentration, then emulsifies it with Alhydrogel aluminum adjuvant and AddVax adjuvant respectively, and then immunizes 6-8 week old female mice in groups. The immunization grouping, immunogen dosage, and adjuvant information for each group are shown in Table 2, with blank cells indicating "none". The immunization strategy is as follows: Figure 2 As shown, each cotton rat was immunized three times via intramuscular injection in the thigh on days 0, 21, and 42, with each injection consisting of 200 μl, 100 μl in each thigh. Blood samples were collected from the orbital fossa on days 19, 40, and 56. After standing, the blood samples were centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then inactivated in a 56°C water bath for 30 minutes and stored at -80°C for specific antibody titer detection and RSV neutralization experiments.

[0054] Table 2. Immunization grouping, immunogen dosage and adjuvant usage in each group.

[0055]

[0056] Example 4: ELISA assay for detecting antigen-induced specific antibody titers

[0057] The RSV novel F antigen dimer protein obtained in Example 1 was diluted to 3 μg / ml with ELISA coating buffer and added to each well of a 96-well ELISA plate (Coring, 3590). After incubation at 4°C for 12 hours, the coating buffer was discarded, and the plate was washed twice with PBS. Then, 100 μl of 5% skim milk powder prepared with PBS was added to each well as blocking buffer, and the plate was blocked at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and 100 μl of serially diluted serum samples obtained in Example 3 were added. Serum samples were serially diluted 4-fold starting from 50-fold with blocking buffer, for a total of 12 dilutions. 100 μl of blocking buffer was added directly to the negative control wells. After incubation at 37°C for 1 hour, the supernatant was discarded, and the plate was washed 3 times with PBST. Then, chicken anti-mouse IgG secondary antibody diluted 1:5000 with blocking buffer was added, and the plate was incubated at 37°C for 1 hour, followed by washing 4 times with PBST. Add 1:5000 dilution of HRP-conjugated goat anti-chicken IgG triple antibody to blocking buffer, incubate at 37°C for 0.5 hours, then wash the plate four times with PBST. Add TMB chromogenic buffer for color development, and stop the reaction with 2M hydrochloric acid after an appropriate reaction time. Detect OD on a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0058] The results are as follows Figure 3 As shown, throughout the immunization process, the level of specific antibodies in the serum of rats in each immunization group increased significantly with the number of immunizations. The highest antibody levels were observed after three immunizations, with the levels observed at 10 μg aluminum adjuvant and 10 μg AddaVax adjuvant. This result indicates that the new RSV F antigen dimer can effectively activate the antibody response in rats.

[0059] Example 5: ELISA assay for the conformation of F neoantigen dimer in vivo

[0060] The RSV novel F antigen dimer protein and RSV post-F protein obtained in Example 1 were diluted to 3 μg / ml with ELISA coating buffer and added to each well of an ELISA 96-well plate (Coring, 3590), 100 μl per well. After incubation at 4°C for 12 hours, the coating buffer was discarded, and the plates were washed twice with PBS. Then, 100 μl of 5% skim milk powder prepared with PBS was added to each well as the blocking buffer, and the plates were blocked at 37°C for 1 hour.

[0061] Serum samples collected after the triple immunization obtained in Example 3 were serially diluted 4-fold starting from 25-fold with blocking buffer, for a total of 12 dilutions. Each diluted sample was then mixed with an equal volume of 6 μg / ml RSVpost-F protein solution prepared with blocking buffer and incubated at 37°C for 1 hour. The mixture was then incubated with 96-well plates coated with RSV novel F antigen dimer protein at 7°C for 1 hour. After discarding the supernatant, the plates were washed three times with PBST. A 1:5000 dilution of chicken anti-mouse IgG secondary antibody with blocking buffer was added, and the plates were incubated at 37°C for 1 hour, followed by four washes with PBST. A 1:5000 dilution of HRP-conjugated goat anti-chicken IgG tertiary antibody with blocking buffer was added, and the plates were incubated at 37°C for 0.5 hours, followed by four washes with PBST. TMB chromogenic buffer was added for color development, and the reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. OD values ​​were then measured using a microplate reader. 450 Read value.

[0062] Serum samples collected after the third immunization, obtained in Example 3, were serially diluted 4-fold starting from 50-fold with blocking buffer, for a total of 12 dilutions. These were then incubated directly at 37°C with 96-well plates coated with RSV post-F protein (washed with PBS). After 1 hour, the supernatant was discarded, and the plates were washed three times with PBST. Then, chicken anti-mouse IgG secondary antibody diluted 1:5000 with blocking buffer was added, and the plates were incubated at 37°C for 1 hour, followed by four washes with PBST. Next, goat anti-chicken IgG tertiary antibody conjugated with HRP was added, and the plates were incubated at 37°C for 0.5 hours, followed by four washes with PBST. TMB chromogenic buffer was added for color development, and the reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. OD values ​​were then measured using a microplate reader. 450 Read value.

[0063] The antibody titer calculation method is the same as in Example 3. Data on specific antibody binding in the triple-immune serum (triple-immune), direct binding between serum and post-F, and competitive binding of serum and post-F to RSV F protein are plotted and analyzed together (e.g., Figure 4 As shown in the figure, the data from the three-immune tests showed a significant difference from the direct data, but no statistical difference from the competing data. This result indicates that the RSV novel F antigen dimer protein exists primarily in the pre-F form in vivo. Although the direct binding data showed that a certain proportion of antibodies in serum can bind to post-F, this is due to the shared epitopes of pre-F and post-F, such as the known epitopes II and IV. There may also be some unknown shared binding antibodies.

[0064] Example 6: Virus neutralization experiment with immune serum

[0065] The serum obtained in Example 3 was initially diluted 1:5, and then serially diluted 4-fold seven times, each diluted with an equal volume of 200 TCID50. 50RSV A2 and RSV B9320 were mixed. After co-incubating at 37°C for 1 hour, 100 μl of the mixture was added to a 96-well plate with approximately 70% HEp-2 cell coverage. After incubating at 37°C for 2 hours, the mixture was discarded, and 100 μl of DMEM medium containing 2% FBS was added. After incubating at 37°C for 4 days, the medium was discarded, the cells were washed twice with PBS, and fixed with ice-cold 80% acetone solution for 20 minutes. The supernatant was discarded, and the cells were washed twice with PBS. 100 μl of 5% skim milk powder prepared with PBS was added to each well, and the cells were blocked at 37°C for 1 hour. After blocking, the blocking solution was discarded, and 100 μl of Palivazumab primary antibody solution with a concentration of 3 μg / ml prepared with the blocking solution was added to each well. After incubating at 37°C for 1 hour, the plate was washed three times with PBST. Add HRP-conjugated goat anti-human IgG secondary antibody diluted 1:5000 with blocking buffer, incubate at 37°C for 1 hour, then wash the plate 4 times with PBST. Add TMB chromogenic buffer for color development, and stop the reaction by adding 2M hydrochloric acid after an appropriate reaction time. Detect OD on a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0066] The results are as follows Figure 5 , Figure 6 As shown, throughout the immunization process, the level of neutralizing antibodies in the serum of mice in each immunization group increased significantly with the number of immunizations. The highest antibody levels were observed after three immunizations at the levels of 10 μg aluminum adjuvant and 3 μg AddaVax adjuvant. This result indicates that the new RSV F antigen dimer can effectively stimulate mice to produce neutralizing antibodies, thereby achieving immune protection.

[0067] Example 7: Cotton Mouse Virus Challenge Protection Experiment

[0068] In Example 3, 10 cotton mice were infected nasally with [the virus] on day 63. 5 TCID 50 RSV A2 virus (such as) Figure 2 (As shown), the animals were euthanized 5 days later, and their lungs, trachea, and nasal bones were removed. The tissue homogenate was used for viral titer (TCID). 50 ) Test. Results are as follows Figure 7 As shown, compared with the PBS control group, the viral load in the tissues of the vaccine-treated mice was significantly reduced, demonstrating a good protective effect.

[0069] Example 8: Protective effect of vaccine on cotton rat tissues

[0070] The lung and tracheal samples from Example 7 were fixed by immersion in 4% paraformaldehyde solution. After sectioning, the lung tissue samples were stained with hematoxylin and eosin to observe lung lesions. The results are as follows... Figure 8 As shown, the PBS group of mice exhibited inflammatory cell infiltration and alveolitis in their lungs. In contrast, the immunized mice, regardless of whether aluminum or AddaVax adjuvant was used, showed significant relief from viral-induced lung damage, exhibiting only mild damage with clearly visible alveoli and minimal inflammatory cell infiltration. This mild lung damage was primarily due to the relatively large single viral dose. These results demonstrate that the RSV neo-F antigen dimer, as a vaccine, can significantly alleviate lung damage caused by RSV A2 virus infection.

Claims

1. A method for preparing respiratory syncytial virus antigen, characterized in that, The respiratory syncytial virus antigen is obtained by expressing the following antigen-encoding gene: The amino acid sequence of the protein encoded by the antigen-encoding gene is shown in SEQ ID NO:4 or SEQ ID NO:

5.

2. The preparation method according to claim 1, characterized in that, A tag sequence and a stop codon coding nucleotide sequence are also added to the 3' end of the antigen-encoding gene.

3. The preparation method according to claim 2, characterized in that, The tag sequence encodes a nucleotide sequence that encodes 6 histidine residues; The nucleotide sequence encoding the stop codon is a single stop codon.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The respiratory syncytial virus antigen encoding gene is expressed in host cells via a recombinant expression vector to obtain the respiratory syncytial virus antigen.

5. The preparation method according to claim 4, characterized in that, The recombinant vector originates from a mammalian cell expression vector, and the host cell is a mammalian cell; the method also includes the steps of collecting cell supernatant after expression and purifying the respiratory syncytial virus antigen.

6. The preparation method according to claim 5, characterized in that, The recombinant vector originates from the mammalian cell expression vector pCAGGS, and the host cell is mammalian 293F cell.

7. The respiratory syncytial virus antigen prepared by the preparation method according to any one of claims 1 to 6.

8. The respiratory syncytial virus antigen as described in claim 7, characterized in that, The amino acid sequence of the respiratory syncytial virus antigen is shown in SEQ ID NO:

5.

9. The gene encoding the respiratory syncytial virus antigen as described in claim 7 or 8.

10. A recombinant expression vector containing the coding gene as described in claim 9.

11. The recombinant expression vector as described in claim 10, characterized in that, The starting vector for the recombinant vector is a mammalian cell expression vector.

12. The recombinant expression vector as described in claim 11, characterized in that, The starting vector for the recombinant vector is the mammalian cell expression vector pCAGGS.

13. A recombinant host cell containing the encoding gene as described in claim 9.

14. The recombinant host cell as described in claim 13, characterized in that, It is a mammalian cell.

15. The recombinant host cell as described in claim 14, characterized in that, It is a mammalian cell, 293F cell.

16. An antigen composition comprising an active ingredient and an adjuvant, wherein the active ingredient is the respiratory syncytial virus antigen as described in claim 8 or 9.

17. The antigen composition according to claim 16, characterized in that, The adjuvant is an aluminum adjuvant or an AddaVax adjuvant.

18. The use of the respiratory syncytial virus antigen as described in claim 7 or 8 in the preparation of a reagent for immunizing animals to obtain RSV-specific antibodies.

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