A bivalent respiratory syncytial virus (RSV) vaccine

Abstract

The application discloses a bivalent respiratory syncytial virus (RSV) vaccine, and relates to the technical field of human vaccines.The vaccine is prepared by mixing RSV-A subtype and RSV-B subtype pre-F trimer proteins and other substances in a certain proportion, and is formulated into a liquid dosage form by adding 0.35 mg / 0.5 mL aluminum hydroxide adjuvant. The p27 fragment and C-terminal esterification region are deleted from the two F proteins, F2 / F1 is connected by GSGSGS, and interchain disulfide bonds are introduced at S146C / N460C and A149C / corresponding Y458C sites, and intrachain disulfide bonds are introduced at S155C / S290C sites. The pre-F trimer is spontaneously assembled into a stable pre-F trimer after being expressed by CHO cells. The purity of the obtained RSV-A antigen is greater than or equal to 98%, and the purity of the RSV-B antigen is 100%. The vaccine does not need to be freeze-dried, and can still maintain high purity and neutralizing epitope integrity after being stored at 37 DEG C for 28 days, can significantly induce neutralizing antibodies and T cell immunity, and is applied to the prevention of RSV-A / B infection of infants, pregnant women in the late stage of pregnancy and the elderly.

Description

Technical Field

[0001] This invention relates to the field of human vaccine technology, and in particular to a bivalent respiratory syncytial virus (RSV) vaccine. 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. RSV was first isolated from chimpanzee respiratory samples in 1956 and subsequently identified as a human pathogen in 1957, exhibiting specificity and pathogenicity in humans. The virus is transmitted through droplets, infecting cells along the human respiratory tract from the nose to the lungs. Initial symptoms of RSV infection 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, although most children may experience mild symptoms during RSV infection... Fever is a common symptom, although a certain percentage of young children may not experience it. 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 as common cold symptoms, but can lead to severe pneumonia in the elderly and those with weakened immune systems. Severe lung disease can lead to low oxygen levels, respiratory muscle fatigue, and sometimes death. Early-life RSV lower respiratory tract infection 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 available 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 (WHO recommends vaccination in late pregnancy, starting in most cases at 28 weeks of gestation). Maternal immunization can transfer antibodies against RSV to unborn infants through the placenta, providing protection for approximately 6 months after birth.

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

[0005] The 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 different prevalence ratios, A accounting for 60-70% and B accounting 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 cope with the virus epidemic. If only the main type A is used, as the immunized population expands, type B will gradually become the dominant strain, which is not conducive to RSV epidemic 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 distinguish 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 mainly comes 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 pre-F and post-F cross-reactive antibodies, 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 diseases; Abrysvo has also demonstrated the ability to protect infants from RSV through maternal vaccination; the monovalent vaccine Arexvy is based on the F protein antigen derived from the A2 strain; Abrysvo is a bivalent vaccine that protects against both subtype A and subtype B RSV infection.

[0009] 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, 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. Summary of the Invention

[0010] In view of the aforementioned existing problems, the present invention is proposed.

[0011] This invention provides a bivalent respiratory syncytial virus (RSV) vaccine that addresses the problems of poor trimer stability requiring freeze-drying, safety concerns due to exogenous sequences, and low purity caused by enzyme digestion residues in existing RSV vaccines.

[0012] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, embodiments of the present invention provide a bivalent respiratory syncytial virus (RSV) vaccine, comprising, RSV-A subtype pre-F trimer protein; RSV-B subtype pre-F trimer protein; Aluminum adjuvant; The vaccine is a liquid formulation, and both pre-F trimer proteins are covalently linked by interchain disulfide bonds to form a stable structure.

[0013] In a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, the pre-F trimer protein monomer is obtained through the following reconstruction: a) Delete the p27 fragment (SEQ No. 3) and the furin restriction site (SEQ No. 4) between the F2 and F1 domains; b) Connect the F2 and F1 structural domains using the GSGSGS connector; c) Delete the C-terminal esterification region SEQ No. 5; d) Replace the N-terminal viral signal peptide with the CHO cell secretion signal peptide SEQ No.8MEWSWVFLFFLSVTTGVHS.

[0014] In a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, the interchain disulfide bonds are formed by the substitution of the following amino acids: In the RSV-A subtype: S146C and N460C; In the RSV-B subtype: A149C and Y458C.

[0015] As a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, it further comprises an intrachain disulfide bond, which stabilizes the pre-F configuration through S155C and S290C mutations.

[0016] As a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, wherein: the CHO cells secrete a signal peptide that causes the pre-F trimer to be secreted into the cell culture medium.

[0017] As a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, wherein the RSV-A subtype pre-F trimer protein has a purity of ≥98% and the RSV-B subtype has a purity of 100%.

[0018] As a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, wherein the aluminum adjuvant is aluminum hydroxide, and each 0.5 mL dose contains 0.35 mg of aluminum.

[0019] As a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine described in this invention, each 0.5 mL dose contains: 60 μg of RSV-A subtype pre-F trimer protein; 60 μg of RSV-B subtype pre-F trimer protein; Aluminum hydroxide, in Al 3+ Total: 0.35 mg; NaCl 135 mmol / L; Tris-HCl 20 mmol / L, pH 7.6; Tween-80 0.5‰.

[0020] In a preferred embodiment of the bivalent respiratory syncytial virus (RSV) vaccine of the present invention, the linker is GSGSGS.

[0021] Secondly, embodiments of the present invention provide the use of the above-mentioned vaccine in the preparation of drugs for preventing RSV infection.

[0022] The beneficial effects of this invention are as follows: By introducing three pairs of interchain disulfide bonds between F protein monomers and simultaneously constructing S155C / S290C intrachain disulfide bonds within the single chain, this invention directly locks the antigen in the pre-F conformation; after an accelerated test at 37°C for 28 days, it still maintains ≥95% trimer purity, which is significantly better than existing products that require freeze-drying.

[0023] 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.

[0024] This invention allows for the production of mature proteins without intracellular enzyme digestion after expression, avoiding residual p27 that could reduce purity or mask antigen sites, and simplifying the purification process.

[0025] 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.

[0026] This 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 also resists immune escape strains of type B, adapting to alternating or co-circulating patterns in different regions.

[0027] In a mouse model, this invention showed that the serum neutralizing antibody GMT induced by the aluminum-containing vaccine was 7-37 times higher than that induced by the aluminum-free antigen; CD4 + / 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.

[0028] 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

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] 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 the present invention.

[0031] Figure 2 This is a graph showing the HPLC purity and trimer retention time of the LZ918 stock solution according to the present invention.

[0032] Figure 3 This is a HPLC purity and trimer retention time analysis chart of the LZ919 stock solution according to the present invention.

[0033] Figure 4 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 according to the present invention.

[0034] Figure 5 This is a comparative analysis of serum antibody GMT in BALB / c mice immunized with different doses of LZ961 antigen without aluminum hydroxide adjuvant according to the present invention.

[0035] Figure 6 This is a pie chart showing the distribution of various cytokines in CD4+ T cells of BALB / c mice after immunization according to the present invention.

[0036] Figure 7 This is a pie chart showing the distribution of various cytokines in CD8+ T cells of BALB / c mice after immunization according to the present invention. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] 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.

[0041] To address the aforementioned technical challenges in the RSV vaccine field, this invention designs a bivalent RSV vaccine containing trimers of the F protein from isotypes A and B. The monomers of each isotype 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.

[0042] For subtype A, this invention conducted point mutation studies, which are discussed in detail in another patent regarding a respiratory syncytial virus pre-F protein trimer and its applications. 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.

[0043] 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 SEQ No. 1 from strain A2 was selected, and for subtype B, the sequence SEQ No. 2 from strain 18537 was selected. The signal peptide and esterification region of both sequences were deleted before being used as the research objects. The BLAST program was run online, 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 No. 2 Isotype B and SEQ 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 their 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.

[0044] The reconstruction for the B subtype is as follows: A segment of the p27 sequence in the middle of the F protein is deleted. The p27 sequence is SEQ No. 3 EAPQYMNYTINTTKNLNVSISKKRKRR. Preferably, the six amino acids at the N-terminal furin restriction site of the p27 sequence (SEQ No. 4 NNRARR) are simultaneously deleted, along with the nine hydrophobic amino acids at the C-terminus (SEQ No. 5 FLGFLLGVG). 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 combinations in this invention are A149C and Y458C, forming a stable pre-F structure. This yields SEQ No. 6, 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.

[0045] By mixing recombinant proteins of subtype A and subtype B according to the method and formulation of this invention and preparing them with aluminum adjuvant, a bivalent RSV vaccine can be obtained. This vaccine can induce humoral and cellular immune responses in animals and exhibits very good immunogenicity.

[0046] Example 1, the first embodiment of the present invention, provides the design of the amino acid sequence of the RSV-A subtype F protein, including: The RSV A2 strain sequence 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 were used as the starting sequence SEQ No. 1. Further deletions were made of amino acids at p27 and furin restriction sites, resulting in the deletion of a total of 42 amino acids. Among them, SEQ No. 4 NNRARR was deleted at the N-terminus, and SEQ No. 5 FLGFLLGVG was additionally deleted at the C-terminus, resulting in SEQ No. 7. To form interchain disulfide bonds, the preferred point mutations are S146C and N460C.

[0047] Example 2 is the second embodiment of the present invention, which provides the design of the amino acid sequence of the RSV-B subtype F protein, including: The sequence of RSV strain 18537 was obtained from the NCBI database. The sequence was adjusted by adding intra-chain disulfide bond mutations S155C and S290C, deleting the N-terminal 25-amino acid signal peptide and the C-terminal palmitoylated sequence, and using this as the starting sequence SEQ No. 2. Further deletion of amino acids at p27 and furin restriction sites was performed, resulting in the deletion of a total of 42 amino acids. Among them, SEQ No. 4 NNRARR was deleted at the N-terminus, and SEQ No. 5 FLGFLLGVG was additionally deleted at the C-terminus, resulting in SEQ No. 6. For the formation of inter-chain disulfide bonds, the preferred point mutations are A149C and Y458C.

[0048] To clearly demonstrate the design schemes of RSV-A and RSV-B subtype mutants in Examples 1 and 2, a summary of the protein mutant design is listed in Table 1.

[0049]

[0050] 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.

[0051] Example 3, the third embodiment of the present invention, provides the expression of recombinant mutants and preliminary cloning screening of antigen expression strains, including: Both RSV-A and RSV-B use the same CHO cell host and the same expression vector, and can be operated by general technicians in the field by following the instructions.

[0052] Prepare 0.8 ml of CHO-K1 cell suspension and 1.5 × 10⁻⁶ CHO-K1 cell suspension. 6Cells / 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, and 30 ml of CHO culture medium was added. The flask was incubated at 37℃ on a 5% CO2 shaker at 136 rpm for 24 hours. Cells were then 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 transferred into 96-well flat-bottomed culture plates using limiting dilution. 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. Then, ELISA was used to check for Ø or III epitopes. RSV-A was preferred for checking Ø epitopes, and RSV-B for checking III epitopes. Positive cells in 24-well and 6-well cell culture plates were screened sequentially. Cells were continuously passaged to establish a cell bank.

[0053] Example 4, refer to Figure 2 and Figure 3 This is the fourth embodiment of the present invention, which provides fermentation and purification of recombinant mutants, including: The RSV-A and RSV-B cell lines screened in Example 3 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 high-purity recombinant protein, i.e., the 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 of LZ918 stock solution are shown in the chromatogram. The retention time is 12.336 min and the purity is 98.6%. Figure 3 The figure shows the HPLC purity and retention time analysis of LZ919 stock solution. The retention time is 10.901 min and the purity is 100%.

[0054] Judging from the retention time, the RSV pre-F mutant of the present invention can form a trimer and is very stable, with a purity of over 98% and no monomer peaks, which fully meets the purity standards for subsequent vaccine preparation.

[0055] Example 5, the fifth embodiment of the present invention, provides a bivalent vaccine preparation and prescription, including: Preparation of the RSV bivalent aluminum adjuvant vaccine: Referring to the dosage design of marketed products, the bivalent vaccine was prepared using our self-made aluminum adjuvant (0.7 mg / ml). The specific steps were as follows: The LZ918 and LZ919 antigen stock solutions were diluted to 240 μg / ml with dilution buffer, then mixed in equal volumes. The mixed bivalent stock solution was slowly added dropwise to an equal volume of aluminum adjuvant suspension, mixing constantly. This bivalent vaccine was named LZ961. Each dose was 0.5 ml. It was aliquoted for subsequent research. The bivalent vaccine formulation is shown in Table 2 below.

[0056]

[0057] Example 6, refer to Figure 4 and Figure 5 This is the sixth embodiment of the present invention, which provides a comparative study of the immunogenicity of a vaccine group containing aluminum adjuvant and an antigen group without aluminum adjuvant, including: 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.

[0058] Preparation of LZ961 vaccine for mouse immunization: Three vials of LZ961 vaccine from Example 5 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 3 below.

[0059]

[0060] Preparation of LZ961 antigen group mouse immunization vaccine: Take 0.125 ml of the LZ961 stock solution (without aluminum adjuvant) from the mixture of subtype A and subtype B antigens in Example 5, 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 4 below.

[0061]

[0062] 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 5 below. 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.

[0063]

[0064] 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 incubated at 37℃ for 60 min. Goat anti-mouse IgG-HRP conjugate was added, and the reaction was incubated at 37℃ for 60 min. TMB chromogenic solution was added for color development, and the reaction was terminated with stop solution. The absorbance was measured (450 nm) 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 6.

[0065]

[0066] 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 at the same time points (D21, D42, D63) after immunization with high-dose LZ961 vaccines (10 μg / mouse) with different aluminum contents were significantly different, except for the group immunized with 10 μg LZ961 vaccine for 21 days. Comparing the GMT of specific antibody titers at the same time points (D21, D42, D63) after immunization with high-dose LZ961 vaccines (10 μg / mouse) with different aluminum contents, the GMT of the 0.35 mg aluminum content group was 7.1, 32.5, and 31.7 times that of the 0 mg aluminum content group, respectively. The GMT of the medium-dose LZ961 vaccines (5 μg / mouse) with different aluminum contents at the same time points (D21, D42, D63) after immunization with medium-dose LZ961 vaccines (5 μg / mouse) with different aluminum contents was also significantly different. 42, D63) Comparison of specific antibody GMT: The GMT of mice in the 0.35mg aluminum group was 18.8, 36.7, and 15.2 times that of the 0mg aluminum group; Comparison of specific antibody GMT at the same time points (D21, D42, D63) after immunization with low-dose LZ961 vaccine (2.5μg / mouse) with different aluminum contents: The GMT of mice in the 0.35mg aluminum group was 16.0, 9.9, and 10.0 times that of the 0mg aluminum group.

[0067] 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 4 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 5 The comprehensive analysis of the above results shows that the selection of an aluminum content of 0.35 mg / 0.5 ml for the LZ961 vaccine is necessary, and the presence of aluminum hydroxide adjuvant in the vaccine can significantly improve the titer of specific antibodies produced by the body.

[0068] 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.

[0069] Example 7, referring to Figure 6 and Figure 7 This is the seventh embodiment of the present invention, which provides a cellular immune response induced by a bivalent RSV vaccine, including: 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.

[0070] 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 count / ml, 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.

[0071] 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 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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 cells expressing six cytokines—IFN-γ, IL-2, TNF-α, CD40L, IL-4, and IL-17—was analyzed. The percentage and number of positive cells expressing each cytokine were calculated using EXCEL. Statistical analysis and variance analysis were performed using GraphPad Prism 8. The expression of four cytokines (IFN-γ, IL-2, TNF-α, and CD40L) in CD4+ or CD8+ T cells were analyzed, and the results are shown in Table 7.

[0077]

[0078] The results above show that in the aluminum hydroxide adjuvant group, after specific stimulation with a mixed RSV-A and RSV-B antigen, CD4+ T cells expressed a small amount of CD40L (10%), and CD8+ T cells expressed a small amount of IL-2 (10%) and CD40L (20%). The positive rates of all cytokines were below 50%. When aluminum hydroxide adjuvant was not present and the LZ961 immunization dose was 2.5 μg / 0.5 ml / animal, after specific stimulation with a mixed RSV-A and RSV-B antigen, CD4+ T cells mainly expressed CD40L (90%), followed by TNF-α (80%), IFN-γ (60%), and IL-2 (40%); CD8+ T cells mainly expressed IFN-γ and CD40L, each accounting for 50%, followed by IL-2 (40%) and TNF-α (20%). When the adjuvant containing aluminum hydroxide was 0.35 mg / 0.5 ml and the LZ961 immunization dose was 2.5 μg / 0.5 ml / animal, after specific stimulation with a mixed RSV-A and RSV-B antigen, the CD4+ T cells mainly expressed TNF-α (100%), followed by IFN-γ (80%), IL-2 (80%), and CD40L (70%); the CD8+ T cells mainly expressed TNF-α (60%), followed by IL-2 and CD40L, each accounting for 50%, and IFN-γ accounted for 20%. When the animal was not treated with aluminum hydroxide adjuvant and the LZ961 immunization dose was 5 μg / 0.5 ml / animal, after specific stimulation with a mixed RSV-A and RSV-B antigen, its CD4+ T cells mainly expressed TNF-α (89%), followed by IL-2 (78%), IFN-γ (44%), and CD40L (44%); its CD8+ T cells mainly expressed CD40L, accounting for 44%, followed by IFN-γ and TNF-α (20%), both accounting for 33%. When the animal was given an adjuvant containing 0.35 mg / 0.5 ml of aluminum hydroxide and an immunization dose of 5 μg / 0.5 ml / animal with LZ961, after specific stimulation with a mixed RSV-A and RSV-B antigen, its CD4+ T cells mainly expressed IFN-γ (90%), followed by IL-2 (80%), CD40L (80%), and TNF-α (70%); its CD8+ T cells mainly expressed IFN-γ (70%), followed by IL-2 (50%), TNF-α (40%), and CD40L (30%).When there is no aluminum hydroxide adjuvant and the LZ961 immunization dose is 10 μg / 0.5 ml / animal, after specific stimulation with mixed RSV-A and RSV-B antigens, its CD4+ T cells mainly express IL-2 (100%), followed by CD40L (70%) and IFN-γ (60%), and TNF-α is expressed the least, at 40%; its CD8+ T cells mainly express IFN-γ (50%), followed by IL-2 (40%), CD40L (40%), and TNF-α (20%). When the adjuvant containing aluminum hydroxide was 0.35 mg / 0.5 ml and the LZ961 immunization dose was 10 μg / 0.5 ml / animal, after specific stimulation with a mixed RSV-A and RSV-B antigen, the CD4+ T cells mainly expressed IL-2 (100%) and TNF-α (100%), followed by IFN-γ (89%) and CD40L (67%); the CD8+ T cells mainly expressed TNF-α (67%), followed by IFN-γ, IL-2, and CD40L, accounting for 56%.

[0079] CD4 + T cells and CD8 + The various cytokines in T cells were plotted and compared separately. Figure 6 CD4 + Pie chart showing the distribution of cytokines in T cells; Figure 7 CD8 + Pie chart showing the distribution of cytokines in T cells; after induction with a mixed RSV-A and RSV-B antigen, RSV-specific T cells in each vaccine group can be divided into the following 5 categories according to the number of cytokine types expressed: CD4+ + or CD8 + The T cell population can be categorized into those expressing four cytokines simultaneously, those expressing three cytokines simultaneously, those expressing two cytokines simultaneously, those expressing only one cytokine to meet the positive criteria, and those expressing no cytokines to meet the positive criteria. Figure 6 and Figure 7 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.

[0080] In summary, the covalent bond connection between the pre-F trimers designed in this invention can remain stable under oxidative conditions, thus eliminating the need for freeze-drying in the final vaccine formulation, making the process simple and convenient. 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. The recombinant viral F protein is expressed using CHO cells as a matrix. Both the A and B subtype F proteins are trimers, exhibiting a pre-F conformation. This trimer has strong structural stability and can induce the production of protective neutralizing antibodies against respiratory syncytial virus and corresponding cellular immune responses in vivo.

[0081] Because this invention uses covalent bonds to form a trimer, 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 structural domains.

[0082] This invention addresses three challenges in the current field of bivalent RSV vaccines and proposes solutions. According to the embodiments of this invention, the aforementioned problems can be satisfactorily resolved. This invention provides a bivalent RSV liquid formulation containing aluminum adjuvant, which does not contain the p27 sequence or the C-terminal exogenous helper sequence. The developed A-type and B-type trimers pre-F exhibit excellent structural stability. Animal experiments show that the vaccine of this invention has very good immunogenicity.

[0083] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A bivalent respiratory syncytial virus (RSV) vaccine, characterized in that, Include: RSV-A subtype pre-F trimer protein; RSV-B isoform pre-F trimer protein; Aluminum adjuvant; The vaccine is a liquid formulation, and both pre-F trimer proteins are covalently linked by interchain disulfide bonds to form a stable structure.

2. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 1, characterized in that, The pre-F trimer protein monomer was obtained through the following reconstruction: a) Delete the p27 fragment (SEQ No. 3) and the furin restriction site (SEQ No. 4) between the F2 and F1 domains; b) Connect the F2 and F1 structural domains using the GSGSGS connector; c) Delete the C-terminal esterification region SEQ No. 5; d) Replace the N-terminal viral signal peptide with the CHO cell secretion signal peptide SEQ No. 8 MEWSWVFLFFLSVTTGVHS.

3. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 2, characterized in that, The interchain disulfide bonds are formed by the substitution of the following amino acids: In the RSV-A subtype: S146C and N460C; In the RSV-B subtype: A149C and Y458C.

4. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 2, characterized in that, It also contains intra-chain disulfide bonds, which stabilize the pre-F configuration through S155C and S290C mutations.

5. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 2, characterized in that, The CHO cell secretion signal peptide causes pre-F trimer to be secreted into the cell culture medium.

6. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 1, characterized in that, The purity of the RSV-A subtype pre-F trimer protein is ≥98%, and the purity of the RSV-B subtype is 100%.

7. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 1, characterized in that, The aluminum adjuvant is aluminum hydroxide, and each 0.5 mL dose contains 0.35 mg of aluminum.

8. The bivalent respiratory syncytial virus (RSV) vaccine as described in claim 1, characterized in that, Each 0.5 mL dose contains: 60 μg of RSV-A subtype pre-F trimer protein; 60 μg of RSV-B subtype pre-F trimer protein; Aluminum hydroxide, in Al 3+ Total: 0.35 mg; NaCl 135 mmol / L; Tris-HCl 20 mmol / L, pH 7.6; Tween-80 0.5‰.

9. A bivalent respiratory syncytial virus (RSV) vaccine as described in claim 2, characterized in that, The connector is GSGSGS.

10. Use of the bivalent respiratory syncytial virus (RSV) vaccine as described in any one of claims 1 to 9 in the preparation of a drug for the prevention of RSV infection.

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