An RSVpre-F trimeric protein combination and its application in the preparation of a bivalent RSV vaccine

Abstract

This invention discloses an RSV pre-F trimer protein combination and its application in the preparation of a bivalent RSV vaccine, relating to the field of human vaccine technology. The vaccine is prepared by mixing equal masses of RSV-A and RSV-B subtype pre-F trimer proteins and adding 0.35 mg / 0.5 mL of aluminum hydroxide adjuvant. Both F proteins have their p27 fragment and C-terminal esterification region deleted, and F2 / F1 are linked by GSGSGS. Interchain disulfide bonds are introduced at S146C / N460C and A149C / Y458C sites, and intrachain disulfide bonds are introduced at S155C / S290C sites. After secretion and expression by CHO cells, the pre-F trimer spontaneously assembles into a stable pre-F trimer. The vaccine does not require lyophilization and maintains high purity and the integrity of neutralizing epitopes after storage at 37°C for 28 days. It can significantly induce neutralizing antibodies and T-cell immunity and can be used to prevent various RSV-A / B infections.

Description

Technical Field

[0001] This invention belongs to the field of human vaccine technology, and in particular relates to an RSV pre-F trimer protein combination and its application in the preparation of bivalent RSV vaccines. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading pathogens causing acute lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. First isolated from chimpanzee respiratory samples in 1956, it was subsequently identified as a human pathogen in 1957, exhibiting specificity and pathogenicity in humans. The virus spreads through droplets, infecting cells along the human respiratory tract from the nose to the lungs. The World Health Organization estimates that approximately 33 million children under the age of five are infected globally each year, with about 3 million requiring hospitalization and 60,000 resulting in death. Initial symptoms typically appear 4–7 days after exposure. In the upper respiratory tract, signs and symptoms include runny nose, sore throat, headache, fatigue, and fever. Notably, while most children may develop fever during RSV infection, a certain percentage of young children may not. Signs and symptoms of lower respiratory tract infection include cough, shortness of breath, rapid breathing, bronchospasm, and wheezing. Severe cases in infants and young children may present with respiratory distress, cyanosis, and feeding difficulties. In adults, RSV often presents with common cold symptoms, but in the elderly and those with weakened immune systems, it can lead to severe pneumonia. Severe lung disease can cause low oxygen levels, respiratory muscle fatigue, and even death. Early-life RSV lower respiratory tract infections can lead to long-term respiratory consequences, including recurrent hospitalizations for respiratory illnesses in infants, recurrent wheezing and asthma attacks, and impaired lung health after infancy.

[0003] Currently approved vaccines are generally used to prevent severe respiratory syncytial virus (RSV) disease in infants and the elderly. To protect infants, vaccines can be administered to pregnant women and those in late pregnancy (the WHO recommends vaccination in late pregnancy, in most cases starting at 28 weeks of gestation). This allows antibodies against RSV to be transferred to the unborn infant through the placenta, providing protection for approximately six months after birth.

[0004] RSV belongs to the Pneumoviridae family and is a single-stranded, negative-sense RNA virus. The viral particles are pleomorphic, ranging from 150 to 300 nm in diameter, with a genome length of 15.2 kb encoding 11 proteins. During infection, RSV causes cells to fuse, forming large cells called syncytia. The virus's structure is primarily composed of three membrane proteins: a small hydrophobic protein (SH), an attachment glycoprotein (G), and a fusion protein (F). The G and F proteins play crucial roles in cell fusion and also stimulate the body to produce protective antibodies. The viral replication cycle is prone to errors, allowing for rapid mutation, which leads to variations in RSV virulence and difficulties in developing antiviral drugs or vaccines.

[0005] The specific prevalence of the two main RSV genotypes varies by region, season, and population. RSV-A is usually associated with more severe clinical symptoms, replicates faster, and may be more likely to cause severe infection. RSV-B has slightly lower pathogenicity, but some strains may have a strong ability to evade the immune system. RSV-A and RSV-B exhibit alternating and co-circulation. Alternating prevalence is dominant, with most regions showing an alternating trend, with A predominating in the first year and B predominating in the following year. In some seasons, both are prevalent, with varying prevalence ratios: A accounts for 60-70%, and B accounts for 30-40%. The prevalence of subtypes A and B also shows regional differences. Some studies in China show that type A is dominant, but RSV vaccines covering both subtypes are more advantageous, creating comprehensive immune pressure to combat the virus. If only the dominant type A is used, as the immunized population expands, type B will gradually become the dominant strain, which is detrimental to RSV disease control.

[0006] Subtypes A and B are primarily based on the G antigen and sequence variations, with G being the most variable gene sequence. These two subtypes can further differentiate and identify several genotypes, including 15 different B genotypes and 9 different A genotypes. RSV has been evolving globally, with new genotypes emerging over the years; the B genotype exhibits greater diversity than RSV A. The RSV F protein is more conserved than the G protein in both A and B genotypes and is a primary target for vaccine development. The F protein differs from the A and B genotypes by only 25 amino acids. The F protein contains antigenic sites shared between pre-fusion and post-fusion conformations, as well as sites unique to each conformation. Six major antigenic sites (Ø, I, II, III, IV, V) are defined on the F protein. Some antigenic sites are expressed in both Pre-F and Post-F conformations, while others are present in only one conformation. Antigenic sites I, II, III, and IV are present in both Pre-F and Post-F conformations and exhibit different affinities. Antigenic sites Ø and V are present only in the Pre-F conformation and have the highest affinity for neutralizing antibodies.

[0007] The RSV neutralizing activity in human serum primarily derives from antibodies targeting the pre-fusion conformation of the F protein, more specifically antigenic sites Ø and V. Immunological analysis showed that over 85% of highly effective antibodies were specific to pre-F. Furthermore, pre-F-specific antibodies were more effective than cross-reactive antibodies against both pre-F and post-F, and also more effective than post-F-specific antibodies. In contrast, no virus-neutralizing activity was observed in G protein-specific serum.

[0008] Two stable pre-F protein-based vaccines, Arexvy and Abrysvo, have been authorized in the United States and Europe to protect older adults from RSV-related lower respiratory tract infections. Abrysvo has also been shown to protect infants from RSV through maternal vaccination. The monovalent Arexvy vaccine is based on the F protein antigen derived from the A2 strain, while Abrysvo is a bivalent vaccine that protects against both subtype A and subtype B RSV infection.

[0009] However, the approved bivalent vaccine Abrysvo still has many areas for improvement. These problems can be summarized as follows: First, the prepared trimer is not stable enough, and the formulation needs to be lyophilized and dissolved in water before injection. Second, in order to achieve the trimer, the vaccine developers introduced a T4 phage sequence foldon at the end of the F protein amino acid to assist in the formation of the trimer. These non-viral exogenous sequences pose a threat to the safety and efficacy of the vaccine. Third, the sequence contains a p27 fragment, which needs to be excised by the furinase expressing the host cell. However, experiments have shown that the excision efficiency of furinase is not 100%, and the residual p27 affects the purity and immunogenicity of the vaccine.

[0010] Therefore, it is essential to develop a novel vaccine that can simultaneously prevent infection by both subtype A and subtype B RSV viruses, while also addressing issues such as poor trimeric stability requiring freeze-drying, safety concerns related to exogenous sequences, and low purity due to enzyme residues. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a novel RSV pre-F trimer protein combination for the first time. Bivalent RSV vaccines prepared using this combination can simultaneously prevent infection by both subtype A and subtype B RSV viruses. It also solves problems such as poor trimer stability requiring freeze-drying, safety concerns related to exogenous sequences, and low purity due to enzyme digestion residues.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] One objective of this invention is to provide an RSV pre-F trimer protein assembly, comprising RSV-A subtype pre-F trimer protein and RSV-B subtype pre-F trimer protein. The preparation method involves extracting a portion of the viral F protein sequence, specifically including the following steps: deleting the N-terminal signal peptide, deleting the C-terminal palmitoylated sequence, deleting the p27 fragment and its N-terminal furin restriction site amino acids and C-terminal hydrophobic amino acids, and linking the F2 and F1 domains using a linker; wherein:

[0014] The intrachain disulfide bond mutation sites of the two RSV pre-F trimer proteins are S155C and S290C;

[0015] The linker sequence of the two RSV pre-F trimer proteins is GSGSGS;

[0016] The interchain disulfide bond mutation sites of the RSV-A subtype pre-F trimer protein are S146C and N460C, and the interchain disulfide bond mutation sites of the RSV-B subtype pre-F trimer protein are A149C and Y458C.

[0017] The signal peptide sequences used to express the two RSV pre-F trimer proteins are shown in SEQ ID NO.8, which are linked to the N-terminus of the truncated and spliced ​​F protein.

[0018] Preferably, the amino acid sequence of the N-terminal furin restriction site is shown in SEQ ID NO.4.

[0019] Preferably, the C-terminal hydrophobic amino acid sequence is shown in SEQ ID NO.5.

[0020] Preferably, the amino acid sequence of the RSV-A subtype pre-F trimer protein is shown in SEQ ID NO.7, and the amino acid sequence of the RSV-B subtype pre-F trimer protein is shown in SEQ ID NO.6.

[0021] The second objective of this invention is to provide the application of any of the RSV pre-F trimer protein combinations described above in the preparation of bivalent RSV vaccines.

[0022] The third objective of this invention is to provide a bivalent RSV vaccine, wherein the bivalent RSV vaccine contains any of the RSV pre-F trimer protein combinations described above.

[0023] Preferably, the preparation method of the bivalent RSV vaccine further includes adding a host cell secretion signal peptide to the deletion position of the N-terminal signal peptide of the two RSV pre-F trimer proteins, constructing an expression vector, expressing the two RSV pre-F trimer proteins in host cells, and secreting the two RSV pre-F trimer proteins in cell culture medium.

[0024] Preferably, the host cell is a CHO cell.

[0025] Preferably, the host cell secreted signal peptide sequence is shown in SEQ ID NO.8.

[0026] Preferably, each 0.5 mL dose of the bivalent RSV vaccine comprises: 55-65 μg of pre-F trimer protein of RSV-A subtype; 55-65 μg of pre-F trimer protein of RSV-B subtype; and aluminum adjuvant, in Al... 3+ The dosage is 0.30~0.40mg.

[0027] More preferably, each 0.5 mL dose of the bivalent RSV vaccine comprises: 60 μg of pre-F trimer protein of RSV-A subtype; 60 μg of pre-F trimer protein of RSV-B subtype; and aluminum adjuvant, in Al... 3+ The total dose is 0.35 mg.

[0028] More preferably, the bivalent RSV vaccine further comprises NaCl, Tris-HCl, and Tween-80; the aluminum adjuvant comprises aluminum hydroxide.

[0029] Most preferably, each 0.5 mL dose of the bivalent RSV vaccine contains:

[0030] 60 μg of RSV-A subtype pre-F trimer protein;

[0031] 60 μg of RSV-B subtype pre-F trimer protein;

[0032] Aluminum hydroxide, in Al 3+ Total: 0.35 mg;

[0033] NaCl 135mmol / L;

[0034] Tris-HCl 20 mmol / L, pH 7.6;

[0035] Tween-80 0.5‰.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. This invention introduces three pairs of interchain disulfide bonds between F protein monomers, while simultaneously constructing S155C / S290C intrachain disulfide bonds within the single chain, directly locking the antigen in the pre-F conformation. Accelerated testing at 37°C revealed that it maintained ≥95% trimer purity after 28 days, significantly superior to existing products requiring lyophilization.

[0038] 2. This invention eliminates non-viral fragments such as T4-foldon and can self-assemble using only natural amino acid mutations, reducing potential immunogenicity and safety risks.

[0039] 3. The present invention allows for the production of mature protein without intracellular enzyme digestion after expression, avoiding residual p27 that could reduce purity or mask antigen sites, and simplifying the purification process.

[0040] 4. The antigen of this invention is long-term stable in 20 mM Tris-HCl / 150 mM NaCl / 0.05% Tween-80 buffer and can be directly reacted with 0.35 mg Al. 3+ / Dosage of aluminum hydroxide is adsorbed into the vaccine, eliminating the need for freeze-drying and reconstitution.

[0041] 5. The present invention provides an equal mass mixture of RSV-A and RSV-Bpre-F trimers, which maintains the strong neutralizing epitopes of highly pathogenic type A strains and resists immune escape strains of type B, adapting to the alternating or co-circulation patterns in different regions.

[0042] 6. In a mouse model, the aluminum-containing vaccine induced serum neutralizing antibody GMT levels 7-37 times higher than those without aluminum antigen, and CD4 levels... + / CD8 + The positive rates of TNF-α and IFN-γ on T cells reached over 70% and 40%, respectively, demonstrating both humoral and cellular immunity.

[0043] 7. This invention has high purity and good safety, and can be used as a candidate vaccine for active immunization of women in late pregnancy or for direct inoculation of the elderly and patients with underlying diseases. Attached Figure Description

[0044] Figure 1 This is a graph showing the amino acid sequence identity data between subtype A (strain A2) and subtype B (strain 18537) in the NCBI Blast online alignment analysis of this invention.

[0045] Figure 2 This is a HPLC purity and trimer retention time analysis chart of the LZ918 stock solution in this invention.

[0046] Figure 3 This is a HPLC purity and trimer retention time analysis chart of the LZ919 stock solution in this invention.

[0047] Figure 4The images show the electrophoretic patterns of intermediates in the LZ918 purification process of this invention (left: non-reduction electrophoresis, right: reduction electrophoresis), where: CD represents affinity chromatography samples; VI represents samples with low pH incubation stability; Q represents anion exchange chromatography samples; PG represents gel molecular sieve samples; VF represents filtered samples; and samples without suffix batch numbers represent the original solution.

[0048] Figure 5 The images show the electrophoretic patterns of intermediates in the LZ919 purification process of this invention (left: non-reduction electrophoresis, right: reduction electrophoresis), where: CD represents affinity chromatography samples; VI represents samples with low pH incubation stability; Q represents anion exchange chromatography samples; PG represents gel molecular sieve samples; VF represents filtered samples; and samples without suffix batch numbers represent the original solution.

[0049] Figure 6 This is a comparative analysis of serum antibody GMT in BALB / c mice immunized with different doses of LZ961 vaccine (0.35 mg / 0.5 ml) containing aluminum, as described in this invention.

[0050] Figure 7 This is a comparative analysis of serum antibody GMT in BALB / c mice immunized with different doses of LZ961 antigen without aluminum hydroxide adjuvant in this invention.

[0051] Figure 8 CD4 in BALB / c mice after immunization in this invention + Pie chart showing the distribution of cytokines in T cells.

[0052] Figure 9 CD8+ in BALB / c mice after immunization in this invention + Pie chart showing the distribution of cytokines in T cells. Detailed Implementation

[0053] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0054] The currently approved bivalent vaccine Abrysvo still has many areas for improvement. These problems can be summarized as follows: First, the prepared trimer is not stable enough, requiring lyophilization and dissolution in water before injection. Second, to achieve the trimer, the vaccine developers introduced a T4 phage sequence foldon at the amino acid terminus of the F protein to assist in trimer formation. These non-viral exogenous sequences pose a threat to the safety and efficacy of the vaccine. Third, the sequence contains a p27 fragment, which needs to be excised by the furinase expressing the host cell. Experiments have shown that the furinase excision efficiency is not 100%, and the residual p27 affects the purity and immunogenicity of the vaccine.

[0055] To address the aforementioned technical problems in the field of RSV vaccines, this invention develops a novel bivalent RSV vaccine containing both A-type and B-type F protein trimers. The monomers of each trimer do not contain the p27 sequence and have had their furin restriction sites deleted. The F2 and F1 domains are linked by a GSGSGS linker. A point mutation is performed on F1, replacing two amino acids with cysteine ​​residues, causing the cysteine ​​linkages between different chains to form disulfide bonds. This results in three pairs of interchain disulfide bonds stabilizing the trimer.

[0056] For subtype A, this invention conducted point mutation studies, which are discussed in detail in another patent on a respiratory syncytial virus pre-F protein trimer. The combination of point mutations can form interchain disulfide bonds, thereby achieving covalent linkage of the pre-F trimer and fundamentally solving the problem of trimer depolymerization. Preferably, the S146C and N460C mutations stabilize the trimer in the pre-F state, without post-F-like structures. ELISA results show that the neutralizing epitope Ø is completely preserved. The trimer antigen structure was challenged using a thermally accelerated assay. The results showed that after 28 days of storage at 37°C, the trimer antigen purity reached over 95%, exhibiting excellent stability. Vaccines prepared using this trimer do not require lyophilization to meet regulatory standards.

[0057] Following the successful construction strategy of subtype A, this invention first performed sequence alignment studies between subtype B and subtype A. For subtype A, the sequence of strain A2 was selected, and for subtype B, strain 18537 was selected. The signal peptide and esterification region of 25 amino acids were deleted from both sequences before they were used as the research objects. The sequences of the two strains after deletion were SEQ ID NO.1 and SEQ ID NO.2, respectively. The two sequences were run online using the BLAST program, and the results are shown below. Figure 1 . Figure 1The amino acid positions shown are those without the 25 signal peptides. The results indicate that the amino acid identity between SEQ ID NO.2 Isotype B and SEQ ID NO.1 Isotype A is 93%. Red amino acids indicate non-identical positions, and dots represent identical positions. Comparing the regions of the five point mutation combinations A149C and Y458C, S150C and N460C, A153C and Y458C, A153C and K461C, and S146C and N460C in Isotype A with the corresponding sequence regions in Isotype B, it can be found that the sequences are identical, meaning that point mutations in Isotype A also apply to Isotype B. Point mutations can also cause the reconstructed Isotype B F protein to form a stable pre-F trimer.

[0058] The reconstruction for the B subtype is as follows: A segment of the F protein sequence p27 (SEQ ID NO.3: EAPQYMNYTINTTKNLNVSISKKRKRR) is deleted. Simultaneously, the six amino acids at the N-terminal furin restriction site (SEQ ID NO.4, NNRARR) and the nine hydrophobic amino acids at the C-terminus (SEQ ID NO.5, FLGFLLGVG) of the p27 sequence are also deleted. The deleted p27 is replaced with the GSGSGS linker. The linker provides a flexible connection between the F2 and F1 peptides, facilitating the binding of the F2 and F1 fragment domains. Any of the five point mutation combinations can be selected; the preferred combination in this invention is A149C and Y458C, forming a stable pre-F structure. This yields SEQ ID NO.6. SEQ ID NO.6 is the single-chain amino acid sequence of the B subtype. Expression of this sequence in CHO cells allows for automatic assembly into a pre-F trimer, which is secreted into the cell culture supernatant. This trimer is linked by interchain disulfide bonds, forming a stable structure.

[0059] The bivalent RSV vaccine can be obtained by mixing the A and B subtype recombinant proteins according to the method and formulation of the present invention and adsorbing them with aluminum adjuvant. This vaccine can induce humoral and cellular immune responses in animals and exhibits very good immunogenicity.

[0060] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0061] Example 1: Design of amino acid sequences for RSV-A isotype F protein and RSV-B isotype F protein

[0062] (1) Design of the amino acid sequence of RSV-A subtype F protein: The sequence of RSV strain A2 was obtained from the NCBI database and adjusted. The intrachain disulfide bond mutation points were S155C and S290C. The N-terminal 25 amino acid signal peptide and the C-terminal palmitoylated sequence were deleted. This was used as the starting sequence SEQ ID NO.1. Further, the amino acids of p27 and its N-terminal furin restriction site and C-terminal hydrophobic amino acid were deleted, for a total of 42 amino acids. Among them, the A subtype p27 sequence is SEQ ID NO.9 (ELPRFMNYTLNNAKKTNVTLSKKRKRR), the N-terminal furin restriction site amino acid was deleted by SEQ ID NO.4 (NNRARR), and the C-terminal hydrophobic amino acid was deleted by SEQ ID NO.5 (FLGFLLGVG), resulting in SEQ ID NO.7. In order to form interchain disulfide bonds, the preferred point mutations are S146C and N460C.

[0063] SEQ ID NO.1 A2

[0064] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARR ELPRFMNYTLNNAKKTNVTLSKKRKRR FLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRG WYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYV SNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0065] SEQ ID NO.7 RSV-A subtype F protein

[0066] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGGSGSCAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSL SNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQ LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCT ASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVCKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0067] (2) Design of the RSV-B subtype F protein amino acid sequence: The sequence of RSV strain 18537 was obtained from the NCBI database. The sequence was adjusted, with intrachain disulfide bond mutations at S155C and S290C. The N-terminal 25 amino acid signal peptide and the C-terminal palmitoylated sequence were deleted, which was used as the starting sequence SEQ ID NO.2. Further, the p27 and its N-terminal furin restriction site amino acids and C-terminal hydrophobic amino acids were deleted, for a total of 42 amino acids. Among them, the p27 sequence of subtype B is SEQ ID NO.3 (EAPQYMNYTINTTKNLNVSISKKRKRR), the N-terminal furin restriction site amino acid was deleted by SEQ ID NO.4 (NNRARR), and the C-terminal hydrophobic amino acid was deleted by SEQ ID NO.5 (FLGFLLGVG), resulting in SEQ ID NO.6. To form interchain disulfide bonds, the preferred point mutations are A149C and Y458C.

[0068] Table 1 presents a summary of the design schemes for the RSV-A and RSV-B subtype mutants.

[0069] Table 1 Summary of Design Schemes for Subtype A and Subtype B Mutants

[0070]

[0071]

[0072] SEQ ID NO.2 18537

[0073] QNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARR EAPQYMNYTINTTKNLNVSISKKRKRR FLGFLLGVGSAIASGIAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTFKVLDLKNYINNRLLPILNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0074] SEQ ID NO.6 F protein of RSV - B subtype

[0075] QNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAAGSGSGSAICSGIAVCKVLHLEGEVNKIKNALLSTNKAVVSLS NGVSVLTFKVLDLKNYINNRLLPILNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQL PIYGVIDTPCWKLHTSPLCTTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCT ASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0076] According to the design in the table above, Nanjing GenScript was commissioned to optimize the codons and synthesize the whole genome to construct the expression vector.

[0077] Example 2 Expression of recombinant mutants and preliminary cloning screening of antigen expression strains

[0078] Both RSV-A and RSV-B use the same CHO cell host and the same expression vector. Note that the CHO cell secretion signal peptide SEQ ID NO. 8 (MEWSWVFLFFLSVTTGVHS) is added at the deletion position of the N-terminal signal peptide of the RSV-A and RSV-B isotype F proteins to construct the complete sequence of the expression vector. The complete construction sequence of isotype A is SEQ ID NO. 10, and the complete construction sequence of isotype B is SEQ ID NO. 11. SEQ ID NO. 10 and SEQ ID NO. 11 are inserted into the expression vector after codon conversion, enabling the secretion of the pre-F trimer into the cell culture medium (Note: Sequences 6-7 are the actual protein expression sequences; the signal peptide is automatically cleaved by the cell during protein secretion). Those skilled in the art can perform the operation according to the instructions.

[0079] SEQ ID NO.10 Protein expression sequence of isotype A containing signal peptide

[0080] MEWSWVFLFFLSVTTGVHSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSGSCAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVCKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0081] Protein expression sequence of subtype B with signal peptide, SEQ ID NO.11

[0082] MEWSWVFLFFLSVTTGVHSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAAGSGSGSAICSGIAVCKVLHLEGEVNKIK NALLSTNKAVVSLSNGVSVLTFKVLDLKNYINNRLLPILNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIK EEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0083] Prepare 0.8 ml of CHO-K1 cell suspension and 1.5 × 10⁻⁶ CHO-K1 cell suspension. 6 Cells / ml) were added sequentially with 20 μg of linearized plasmid and transfection reagent. The Gene Pulser Xcell (Bio-Rad) generator was set to 300V, 900μF single pulse, infinite resistance, and a 4mm gap disposable electroporation cuvette (Bio-Rad). The electroporation time was 12-20ms. Cells were transferred from the electroporation cuvette to a culture flask, 30 ml of CHO culture medium was added, and the flask was incubated at 37℃ on a 5% CO2 shaker at 136 rpm for 24 hours. After incubation, cells were collected by low-speed centrifugation at 800 rpm and replaced with a solution containing 50 μM... MSX's CHO culture medium (glutamine-free) was used for pressure screening. Cells were then transferred into 96-well flat-bottomed culture plates using a limiting dilution method. The culture plates were incubated at 37°C in a 5% CO2 incubator. The cells were observed under an inverted microscope, and the wells containing monoclonal cells were labeled. Subsequently, Ø or III epitopes were examined using ELISA. RSV-A was preferred for examining Ø epitopes, and RSV-B for examining III epitopes. Positive cells from 24-well and 6-well cell culture plates were screened sequentially. Cells were continuously passaged to establish two cell banks: type A (code: LZ918) and type B (code: LZ919).

[0084] Gene stability analysis: Type A and Type B cells were thawed in a 37°C water bath. The cell suspension in the cryopreservation tube was transferred to a centrifuge tube containing 5 ml of CD CHO culture medium. After centrifugation at 800 rpm for 10 minutes, the supernatant was discarded, and 5 ml of CD CHO culture medium was added to resuspend the cells in the centrifuge tube. Then, 5 ml of cell suspension was transferred to a 125 ml culture flask containing 25 ml of CD CHO culture medium and cultured at 37°C and 5% CO2 with shaking at 136 rpm. This was recorded as passage 1. After 5 days of culture, the cells were cultured at a density of 50 × 10⁶ cells / year. 4 Cells / ml were transferred to 125ml cell culture flasks containing 25ml of CD CHO culture medium and cultured at 37℃, 5% CO2, and shaken at 136rpm for 4 days. The cells were then passaged again in 125ml flasks, and this process was repeated, with passages every 4 days. A total of 15 passages were performed. During this period, cell morphology was observed; the cells were round, with neat edges, and appeared singly, bright, and in suspension. Cell counts were performed before each passage to determine the starting cell seeding ratio for the next generation, maintaining a constant seeding density of 50 × 10⁶ cells / ml. 4 Cells / ml. Cell viability was above 90% at each passage, with stable cell viability. The doubling time during the logarithmic growth phase was approximately 23 to 31 hours throughout the passage period. Real-time PCR was used to detect the gene copy number of cells from the original seed bank, master seed bank, working seed bank, and different passages of cells cultured in shake flasks during continuous passage in 125 ml cell culture flasks.

[0085] The results showed that the copy number of LZ918 cells remained relatively constant throughout the 15th generation after resuscitation, with a mean copy number of 2.19 copies / cell and an SD value of 0.32 copies / cell, and the cells were stable in the fermentation cells (see Table 2). Similarly, the copy number of LZ919 cells remained relatively constant throughout the 15th generation after resuscitation, with a mean copy number of 2.31 copies / cell and an SD value of 0.52 copies / cell, and the cells were stable in the fermentation cells (see Table 3).

[0086] Table 2. Copy number stability of the target gene in LZ918 cells expressing type A protein after passage.

[0087]

[0088] Formula for calculating the genome copy number of 2ng CHO cells:

[0089] CHO cell genome size: 2.45GB, one genome mass:

[0090] 2.45×10 9 ×650 / 6.02×10 23 =2.645×10 -12 g

[0091] 2ng CHO cell genome copy number = 2 × 10 -9 / 2.645×10 -12 =756

[0092] Cell copy number = Sample copy number / Genome copy number

[0093] Table 3. Copy number stability of the target gene in LZ919 cells expressing type B protein after passage.

[0094]

[0095] Example 3 Fermentation and purification of recombinant mutants

[0096] The RSV-A and RSV-B cell lines screened in Example 2 were cultured in shake flasks according to conventional CHO cell fermentation techniques. After fermentation, the cell fermentation broth was first clarified by centrifugation. The clarified broth was then subjected to CaptoDeVirS gel chromatography to remove most of the host cell proteins and nucleic acids. Following this, it underwent further purification steps including strong anion exchange chromatography, gel filtration chromatography, and weak anion exchange chromatography to remove residual host cell proteins and nucleic acids and pyrogens, yielding a high-purity recombinant protein stock solution. The RSV-A and RSV-B stock solutions had the same formulation and were stored using the following formula: 20 mM Tris-HCl (containing 150 mM NaCl, 0.5‰ Tween 80). The RSV-A protein antigen was named LZ918, and the RSV-B protein antigen was named LZ919. Samples were taken for HPLC purity and retention time analysis; the results are shown below. Figure 2 and Figure 3 . Figure 2 The HPLC purity and trimer retention time analysis of LZ918 stock solution are shown in the chromatogram, which indicates a retention time of 12.336 min and a purity of 98.6%. Figure 3 The figure shows the HPLC purity and retention time analysis of the LZ919 stock solution. The retention time is 10.901 min, and the purity is 100%. Based on the retention time, it can be determined that the RSV pre-F mutant of this invention can form a trimer and is very stable, with a purity of over 98% and no monomer peaks, fully meeting the purity standards for subsequent vaccine preparation.

[0097] The intermediates from the above purification process were analyzed by reducing and non-reducing electrophoresis. See details below. Figure 4 and Figure 5Electrophoresis results of purified intermediates LZ918 and LZ919 showed that the trimer was stable in non-reduction electrophoresis, with no monomers or dimers, while reduction electrophoresis showed that the monomer purity was very high and there were no multimers.

[0098] Example 4: Bivalent Vaccine Preparation and Prescription

[0099] Preparation of the RSV bivalent aluminum-adjuvanted vaccine: First, dilute the LZ918 and LZ919 antigen stock solutions prepared in Example 3 to 240 μg / ml with dilution buffer, then mix them in equal volumes. Slowly add the mixed bivalent stock solution dropwise to an equal volume of aluminum adjuvant suspension, mixing constantly to obtain the bivalent vaccine, which is named LZ961. Each dose is 0.5 ml. It is aliquoted for subsequent research. The specific formulation of the bivalent vaccine is shown in Table 4.

[0100] Table 4. Prescriptions for Bivalent RSV Liquid Vaccines with Aluminum Adjuvant

[0101]

[0102] Example 5: Comparison of immunogenicity between aluminum-adjuvanted vaccine group and aluminum-free antigen group

[0103] BALB / c mice were immunized with bivalent vaccines containing and without aluminum adjuvants to evaluate the immunogenicity of the bivalent vaccines and the necessity of using aluminum adjuvants.

[0104] Preparation of LZ961 vaccine for mouse immunization: Three vials of LZ961 vaccine from Example 4 were randomly selected and mixed thoroughly. 0.25 ml of the mixed vaccine was taken and 11.75 ml of aluminum hydroxide adjuvant solution with an aluminum content of 0.7 mg / ml was added, resulting in a protein content of 2.5 μg / 0.5 ml after dilution. 0.5 ml of the mixed vaccine was taken and 11.5 ml of aluminum hydroxide adjuvant solution with an aluminum content of 0.7 mg / ml was added, resulting in a protein content of 5 μg / 0.5 ml after dilution. 1 ml of the mixed vaccine was taken and 11 ml of aluminum hydroxide adjuvant solution with an aluminum content of 0.7 mg / ml was added, resulting in a protein content of 10 μg / 0.5 ml after dilution. Detailed preparation information is shown in Table 5 below.

[0105] Table 5. Vaccine preparation regimens for mouse immunogenicity studies

[0106]

[0107] Preparation of LZ961 antigen group mouse immunization vaccine: Take 0.125 ml of the LZ961 stock solution (without aluminum adjuvant) from Example 4, add 11.875 ml of dilution buffer, and the protein content after dilution is 2.5 μg / 0.5 ml; take 0.25 ml of LZ961 stock solution, add 11.75 ml of dilution buffer, and the protein content after dilution is 5 μg / 0.5 ml; take 0.5 ml of LZ961 stock solution, add 11.5 ml of dilution buffer, and the protein content after dilution is 10 μg / 0.5 ml. Detailed preparation information is shown in Table 6 below.

[0108] Table 6. Antigen preparation regimens for mouse immunogenicity studies

[0109]

[0110] Seventy animals were randomly divided into seven groups of ten each. Immunization was administered via intraperitoneal injection at a dose of 0.5 ml per animal. Details are shown in Table 7. Immunization: Second immunizations were given on days 21 and 42 after the first immunization, for a total of three immunizations. Blood Collection: Blood samples were collected from the tail vein of mice during the first and second immunizations. Blood was collected from the eyeballs during the third immunization, along with the spleen. Blood collection time points: Blood was collected three times, on days 21, 42, and 63 after the first immunization. Blood samples were placed in 1.5 ml centrifuge tubes, incubated at room temperature for 2 hours, then transferred to 2–8°C overnight, centrifuged at 5000 rpm for 5 minutes, and the separated serum was stored at -20°C or below.

[0111] Table 7. Mouse Immunization Groups and Animal Numbers

[0112]

[0113] ELISA method for detecting antibody titers: RSV type A and type B antigens were coated on blank ELISA plates. Specifically, a mixed antigen (obtained by mixing RSV-A and RSV-B antigens with equal protein content) was used as the coating antigen. The mixed antigen was diluted to 200 ng / ml and added to each well of a corresponding 96-well ELISA plate, 100 μl / well, and incubated overnight at 2-8℃. Pre-diluted mouse serum was added, and the reaction was carried out at 37℃ for 60 min. Goat anti-mouse IgG-HRP conjugate was added, and the reaction was carried out at 37℃ for 60 min. TMB chromogenic solution was added for color development, and the reaction was terminated with stop solution. The absorbance (450 nm) was measured using an ELISA reader. The cutoff value was defined as three times the mean absorbance of the negative control (aluminum hydroxide adjuvanted mouse serum) (if the absorbance was less than 0.1, it was calculated as 0.1). The maximum dilution corresponding to a mean absorbance of the immunized mouse serum higher than the cutoff value was the antibody titer of the immunized mouse serum. The results are shown in Table 8.

[0114] Table 8 Serum GMT antibody after immunization of BALB / c mice

[0115]

[0116] The results above show that the specific antibody titers after immunization with the six LZ961 vaccines (0.35 mg / mouse and 0 mg / mouse with aluminum content) increased with increasing LZ961 vaccine dosage and number of immunizations. For the two LZ961 vaccines with different aluminum contents, at the same LZ961 immunization dose, the specific antibody titers produced by the same LZ961 dosage showed significant differences, except for the group immunized with 10 μg LZ961 vaccine for 21 days. Specifically:

[0117] (1) Comparison of specific antibody titers (GMT) at the same time points (D21, D42, D63) after immunization with high-dose LZ961 vaccine (10μg / mouse) with different aluminum contents: The GMT of mice in the group with aluminum content of 0.35mg was 7.1, 32.5, and 31.7 times that of the group with aluminum content of 0mg;

[0118] (2) Comparison of specific antibody titers (GMT) at the same time points (D21, D42, D63) after immunization with medium-dose LZ961 vaccine (5μg / mouse) with different aluminum contents: The GMT of mice in the group with aluminum content of 0.35mg was 18.8, 36.7, and 15.2 times that of the group with aluminum content of 0mg;

[0119] (3) Comparison of specific antibody titers (GMT) at the same time points (D21, D42, D63) after immunization with low-dose LZ961 vaccine (2.5 μg / mouse) with different aluminum contents: The GMT of mice in the group with aluminum content of 0.35 mg was 16.0, 9.9, and 10.0 times that of the group with aluminum content of 0 mg.

[0120] Serum antibody GMT analysis of BALB / c mice immunized with LZ961 vaccine at various doses (0.35 mg / 0.5 ml) with aluminum content is shown in the figure. Figure 6 Serum antibody GMT analysis of BALB / c mice immunized with each dose of LZ961 vaccine without aluminum hydroxide adjuvant is shown in [reference needed]. Figure 7 .

[0121] The comprehensive analysis of the above results shows that it is necessary to select an aluminum content of 0.35 mg / 0.5 ml for the LZ961 vaccine. The presence of aluminum hydroxide adjuvant in the vaccine can significantly improve the titer of specific antibodies produced by the body.

[0122] No anti-RSV antibodies were found in the serum of the control group mice. Throughout the experiment, no anti-RSV antibodies were detected in the serum of the control group mice (aluminum hydroxide adjuvant), indicating that the animals were not exposed to contaminants that could interfere with the experimental results during the entire experiment.

[0123] Example 6: Cellular Immune Response Induced by Bivalent RSV Vaccine

[0124] Preparation of solutions: Prepare RPMI 1640 cell culture medium, adjust the pH to 7.2-7.4, and store at 2-8℃ for later use. Cell culture medium (RPMI 1640 cell culture medium containing 10% FBS), for later use. Staining buffer (physiological saline containing 1% FBS), prepare fresh before use. Perm / Wash Buffer (1x), prepare fresh before use. Mixed antigen solution (200μg / ml): Take 0.86ml of LZ918 stock solution (4.62mg / ml) and 2.61ml of LZ918 stock solution (1.53mg / ml), add to 36.53ml of cell culture medium, mix well, and prepare fresh before use.

[0125] Staining Procedure: Isolate mouse spleen lymphocytes. Obtain the spleen and grind the lymphocytes. Mix the ground spleen lymphocytes into a suspension and centrifuge at 2500 rpm for 15 min. The centrifuge tube will separate into three layers from top to bottom; the second layer is the lymphocyte layer. Adjust the cell density to approximately 4.0 × 10⁻⁶ cells based on the cell count results. 6 cells / ml, for later use. Ten randomly selected aliquots with a density adjusted to 4.0 × 10⁻⁶ cells / ml were used. 6 For samples with a cell / ml ratio, 900 μl of cell suspension was taken from each sample and added to the same 15 ml centrifuge tube. The mixture was then pipetted and aspirated to obtain mixed mouse lymphocytes for later use. The counted lymphocytes from each test sample were then added sequentially to the corresponding wells of a 24-well cell culture plate at 500 μl / well, with two wells for each test sample. The plates were incubated at 37.0°C for at least 1 hour. 500 μl of mixed antigen (200 μg / ml) was added to well 1 of each test sample as the stimulated sample; 500 μl of cell culture medium was added to well 2 as the unstimulated sample. The 24-well cell culture plate was gently mixed and incubated at 37.0°C for 20 hours in a carbon dioxide incubator containing 5.0% CO2.

[0126] Add inhibitor: Add 1 μl of Protein Transport Inhibitor (containing Brefeldin A) to each well of the sample to be tested. Incubate at 37.0℃ for 4 h in a 5.0% CO2 incubator. Add 2 μl of Leukocyte Activation Cocktail with BD GolgiPlug™ to each well of the single positive control sample. No treatment is required for the blank control sample. Collect cells, block the Fc receptor, and block the non-specific staining caused by the fluorescent antibody Fc receptor with Mouse BD Fc Block™, which specifically targets the Fcγ receptor. Add 100 μl of Mouse BD Fc Block™ pre-diluted 1:50 with Staining Buffer to each tube, vortex to mix, and incubate at 2–8℃ for 15 min. After incubation, add 1 ml of Staining Buffer to each tube of cells to wash the cells, centrifuge at 3500 rpm for 3 min, discard the supernatant, and gently tap the centrifuge tube to suspend the cells. Add 1 ml of Staining Buffer to each tube of cells to wash the cells, centrifuge at 3500 rpm for 3 min, discard the supernatant, and gently tap the centrifuge tube to suspend the cells.

[0127] Cell surface staining: Add 50 μl Brilliant Stain Buffer and 1 μl CD3-BV510, 1 μl CD4-FITC, 1 μl CD8-APC-Cy7, and 1 μl CD40L-BV650 to each tube of cells; vortex to mix and incubate at 2–8°C for 30 min.

[0128] Single-positive control and blank control samples: Add 50 μl of Brilliant Stain Buffer to each tube of cells in the single-positive control. For CD3 single-positive tubes, CD4 single-positive tubes, CD8 single-positive tubes, and CD40L single-positive tubes, add 1 μl of CD3-BV510, 1 μl of CD4-FITC, 1 μl of CD8-APC-Cy7, and 1 μl of CD40L-BV650, respectively. Vortex mix and incubate at 2-8℃ for 30 min.

[0129] After washing, fixing, and rupturing the membrane, intracellular factors were stained: 50 μl Perm / Wash Buffer (1×) and 1 μl IFN-γ-BV421, 1 μl IL-2-PE-Cy7, 1 μl IL-4-APC, 1 μl TNF-α-PE, and 1 μl IL-17-BV421 were added to each tube of cells; after vortexing, the cells were incubated at 2–8°C for 30 min. Single-positive control and blank control samples: Add 50 μl of Perm / Wash Buffer (1×) to each tube of cells in the single-positive control. For the IFN-γ single-positive tubes, IL-2 single-positive tubes, IL-4 single-positive tubes, TNF-α single-positive tubes, and IL-17 single-positive tubes, add 1 μl of IFN-γ-BV421, 1 μl of IL-2-PE-Cy7, 1 μl of IL-4-APC, 1 μl of TNF-α-PE, and 1 μl of IL-17-BV421, respectively. Incubate at 2–8°C for 30 min. For the blank control sample, add 50 μl of Perm / Wash Buffer (1×) and incubate at 2–8°C for 30 min.

[0130] Cytokine detection: Flow cytometer (BD FACSCanto™ Flow Cytometer), data acquisition software (FACS Diva 8.0.1), channel parameters selected: FSC, SSC, FITC, PE, PE-Cy7, APC, APC-Cy7, V450 (BV421), BV650, V500 (BV510), RB705 (Percp-Cy5.5), sample flow rate: medium speed, samples to be tested were acquired, and the number of events to stop acquisition was CD3. + 100,000 events were recorded within the gate. Data was promptly saved after collection and further data processing and analysis were performed.

[0131] Data processing: FlowJo software was used to statistically analyze 100,000 CD3 samples. + CD4 in T cells + T cell in the gate or CD8 + The number of T-cell positive cells expressing six cytokines—IFN-γ, IL-2, TNF-α, CD40L, IL-4, and IL-17—was calculated using Excel. Statistical analysis and variance analysis were performed using GraphPad Prism 8. (The text also mentions CD40L, IL-4, and IL-17, but these are not directly related to the main point about data processing.) + or CD8 + The expression of four cytokines (IFN-γ, IL-2, TNF-α, and CD40L) in T cells was analyzed, and the results are shown in Table 9.

[0132] Table 9 CD4 +and CD8 + Statistical table of T cells with positive cytokines of various types

[0133]

[0134] Note: N = number of mice analyzed, % = positive rate.

[0135] The results above show that in the aluminum hydroxide adjuvant group, after specific stimulation with a mixed RSV-A and RSV-B antigen, CD4 levels were significantly reduced. + T cells expressed low levels of CD40L (10%) and CD8. + T cells expressed small amounts of IL-2 (10%) and CD40L (20%), and the positivity rates of all cytokines were below 50%.

[0136] When the animal was not treated with aluminum hydroxide adjuvant and the LZ961 immunization dose was 2.5 μg / 0.5 ml / animal, its CD4 count increased after specific stimulation with a mixed RSV-A and RSV-B antigen. + T cells primarily express CD40L (90%), followed by TNF-α (80%), IFN-γ (60%), and IL-2 (40%); their CD8+ expression is also significant. + T cells mainly express IFN-γ and CD40L, each accounting for 50%, followed by IL-2 (40%) and TNF-α (20%).

[0137] When the dose of LZ961 immunization was 2.5 μg / 0.5 ml / animal, containing 0.35 mg / 0.5 ml of aluminum hydroxide adjuvant, and the specific stimulation of the mixed RSV-A and RSV-B antigens, the CD4 count was significantly increased. + T cells primarily express TNF-α (100%), followed by IFN-γ (80%), IL-2 (80%), and CD40L (70%); their CD8+ expression is also significant. + T cells mainly express TNF-α (60%), followed by IL-2 and CD40L, each accounting for 50%, and IFN-γ accounting for 20%.

[0138] When the animal was not treated with aluminum hydroxide adjuvant and the LZ961 immunization dose was 5 μg / 0.5 ml / animal, its CD4 count increased after specific stimulation with a mixed RSV-A and RSV-B antigen. + T cells primarily express TNF-α (89%), followed by IL-2 (78%), IFN-γ (44%), and CD40L (44%); their CD8+ expression is also significant. + T cells mainly express CD40L, accounting for 44%, followed by IFN-γ and TNF-α (20%), each accounting for 33%.

[0139] When the dose of LZ961 immunization was 5 μg / 0.5 ml with 0.35 mg / 0.5 ml containing aluminum hydroxide adjuvant, and the specific stimulation of the animal with a mixed RSV-A and RSV-B antigen, its CD4 count increased. + T cells primarily express IFN-γ (90%), followed by IL-2 (80%), CD40L (80%), and TNF-α (70%); their CD8+ expression is also significant. + T cells mainly express IFN-γ (70%), followed by IL-2 (50%), TNF-α (40%), and CD40L (30%).

[0140] When the animal was not treated with aluminum hydroxide adjuvant and the LZ961 immunization dose was 10 μg / 0.5 ml / animal, its CD4 count increased after specific stimulation with a mixed RSV-A and RSV-B antigen. + T cells primarily express IL-2 (100%), followed by CD40L (70%) and IFN-γ (60%), with TNF-α expression being the lowest at 40%; their CD8+ expression is also low. + T cells mainly express IFN-γ (50%), followed by IL-2 (40%), CD40L (40%), and TNF-α (20%).

[0141] When the dose of LZ961 immunization was 10 μg / 0.5 ml with 0.35 mg / 0.5 ml containing aluminum hydroxide adjuvant, and the specific stimulation of the animal with mixed RSV-A and RSV-B antigens, its CD4 count increased. + T cells primarily express IL-2 (100%) and TNF-α (100%), followed by IFN-γ (89%) and CD40L (67%); their CD8+ expression is also significant. + T cells mainly express TNF-α (67%), followed by IFN-γ, IL-2, and CD40L, accounting for 56%.

[0142] CD4 + T cells and CD8 + The various cytokines in T cells were compared and plotted separately, including CD4. + The pie chart showing the distribution of various cytokines in T cells is as follows: Figure 8 As shown, CD8 + The pie chart showing the distribution of various cytokines in T cells is as follows: Figure 9 As shown. The results showed that after induction with a mixed RSV-A and RSV-B antigen, RSV-specific T cells in each vaccine group could be divided into the following 5 categories according to the amount of cytokine expressed: CD4 + or CD8 +The T cell population exhibits the following positive criteria: simultaneous expression of four cytokines, simultaneous expression of three cytokines, simultaneous expression of two cytokines, expression of only one cytokine, and expression of no cytokines meeting the positive criteria. Figure 8 and Figure 9 The results showed that cytokine expression levels and combinations were strongly positively correlated with humoral response data, supporting the technical route of aluminum adjuvant liquid vaccines.

[0143] In summary: (1) The pre-F trimers designed in this invention are covalently linked and can remain stable under oxidative conditions. Therefore, the final vaccine formulation does not require freeze-drying, and the process is simple and convenient. (2) This invention provides a liquid-form bivalent respiratory syncytial virus (RSV) vaccine. The antigens used in this vaccine are derived from circulating strains of RSV subtypes A and B, respectively. CHO cells are used as the matrix to express recombinant viral F protein. Both subtypes A and B F proteins are trimers, exhibiting a pre-F configuration. These trimers have strong structural stability and can induce protective neutralizing antibodies and corresponding cellular immune responses against respiratory syncytial virus in vivo. (3) Because this invention uses covalently linked trimers, there is no need to add other exogenous auxiliary sequences, thus improving the safety of the vaccine. This invention directly deletes the p27 sequence when reconstructing the antigen sequence and uses a flexible linker to connect the two domains. (4) This invention proposes solutions to three challenges in the current field of bivalent RSV vaccines. According to the embodiments of this invention, the above problems can be satisfactorily solved. This invention provides a bivalent RSV liquid formulation containing aluminum adjuvant vaccine, which does not contain the p27 sequence or the C-terminal exogenous helper sequence. The developed A-type and B-type trimers pre-F have excellent structural stability. Animal experiments show that the vaccine of this invention has very good immunogenicity.

[0144] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An RSV pre-F trimer protein combination, characterized in that, This includes RSV-A and RSV-B pre-F trimer proteins, prepared by extracting a portion of the viral F protein sequence, specifically including the following steps: deleting the N-terminal signal peptide, deleting the C-terminal palmitoylated sequence, deleting the p27 fragment and its N-terminal furin restriction site amino acids and C-terminal hydrophobic amino acids, and linking the F2 and F1 domains using a linker; wherein: The intrachain disulfide bond mutation sites of the two RSV pre-F trimer proteins are S155C and S290C; The linker sequence of the two RSV pre-F trimer proteins is GSGSGS; The interchain disulfide bond mutation sites of the RSV-A subtype pre-F trimer protein are S146C and N460C, and the interchain disulfide bond mutation sites of the RSV-B subtype pre-F trimer protein are A149C and Y458C. The signal peptide sequences used to express the two RSV pre-F trimer proteins are shown in SEQ ID NO.8, which are linked to the N-terminus of the truncated and spliced ​​F protein; The amino acid sequence of the RSV-A subtype pre-F trimer protein is shown in SEQ ID NO.7, and the amino acid sequence of the RSV-B subtype pre-F trimer protein is shown in SEQ ID NO.

6.

2. The RSV pre-F trimer protein combination according to claim 1, characterized in that, The amino acid sequence of the N-terminal furin restriction site is shown in SEQ ID NO.

4.

3. The RSV pre-F trimer protein combination according to claim 2, characterized in that, The C-terminal hydrophobic amino acid sequence is shown in SEQ ID NO.

5.

4. The use of the RSV pre-F trimer protein combination according to any one of claims 1 to 3 in the preparation of a bivalent RSV vaccine.

5. A bivalent RSV vaccine, characterized in that, It includes the RSV pre-F trimer protein combination as described in any one of claims 1 to 3.

6. The bivalent RSV vaccine according to claim 5, characterized in that, Each 0.5 mL dose of the bivalent RSV vaccine contains: 55-65 μg of pre-F trimer protein of RSV-A subtype; 55-65 μg of pre-F trimer protein of RSV-B subtype; aluminum adjuvant, in Al 3+ The dosage is 0.30~0.40mg.

7. The bivalent RSV vaccine according to claim 6, characterized in that, The bivalent RSV vaccine also contains NaCl, Tris-HCl, and Tween-80; the aluminum adjuvant includes aluminum hydroxide.

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