A reconstituted respiratory syncytial virus f protein trimer

Abstract

The application discloses a reconstructed respiratory syncytial virus F protein trimer, and relates to the technical field of genetic engineering vaccines.The application designs a RSV F protein trimer, the monomer of the trimer does not contain a p27 sequence, and a furin enzyme cleavage peptide segment aa110-aa136 is deleted; a GS linker is used for connecting between an F2 domain and an F1 domain; the application also designs point mutation combinations, the point mutation combinations can form inter-chain disulfide bonds, so that covalent connection of the trimer is realized, and the problem of trimer depolymerization is fundamentally solved; the trimer is stably kept in a pre-F state, and there is no post-F-like structure; an ELISA result shows that neutralizing epitopes are completely retained; a thermal acceleration experiment method is used to challenge the trimer antigen structure, and the result shows that the trimer antigen purity reaches more than 95% after being placed at 37 DEG C for 28 days, and the stability is very good; the trimer is used to prepare a vaccine, and the vaccine can meet the standard of a regulation requirement without freeze drying.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering vaccine, and particularly relates to a restructured respiratory syncytial virus F protein trimer. BACKGROUND

[0002] Respiratory syncytial virus (RSV) is one of the main pathogens causing acute lower respiratory tract infection in infants, the elderly and immunocompromised persons worldwide; RSV virus was first isolated from the respiratory tract samples of chimpanzees in 1956, and then confirmed as a human pathogen in 1957, which is specific and pathogenic to humans; the virus is transmitted by droplets, from the nose to the lungs along the human respiratory tract to infect cells; the initial symptoms of RSV infection usually appear within 4-7 days after exposure to the virus; in the upper respiratory tract, the signs and symptoms of the disease include: runny nose, sore throat, headache, fatigue and fever; it is worth noting that although most children may have fever symptoms 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 sound; in infants, severe cases can have respiratory distress, cyanosis, feeding difficulties and other manifestations; in adults, RSV often presents as common cold symptoms, and in the elderly and immunocompromised persons, it can cause severe pneumonia; severe lung disease can lead to low oxygen content in the body, respiratory muscle fatigue, and sometimes death; RSV lower respiratory tract infection in early life can lead to long-term respiratory consequences, including repeated hospitalization of infants due to respiratory disease, recurrent wheezing and asthma, and impaired lung health after infancy;

[0003] There is no specific treatment for respiratory syncytial virus disease; the management of severe respiratory syncytial virus disease involves supportive care, such as suctioning nasal secretions, intravenous fluids to replace fluids, and most importantly, oxygen to help breathing; there are currently vaccines available to prevent severe respiratory syncytial virus disease in infants and the elderly; to protect infants, the vaccine can be given to pregnant women and people in the third trimester of pregnancy (WHO recommends vaccination in the third trimester, which in most cases starts at 28 weeks of gestation); maternal immunization can transfer anti-respiratory syncytial virus antibodies to the unborn baby through the placenta, then provide protection to the baby for about 6 months after birth.

[0004] RSV belongs to the Pneumoviridae family and is a single-stranded negative-sense RNA virus; the viral particles exhibit pleomorphism with a diameter of 150-300 nm, and the genome is 15.2 kb long, encoding 11 proteins; when infection develops, RSV causes cell fusion to 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), of which the G protein plays an important role in cell fusion and is also the protein that stimulates the body to produce protective antibodies; the replication cycle of the virus is prone to errors, allowing rapid mutations, which leads to changes in RSV virulence and difficulties in developing antiviral drugs or vaccines;

[0005] Subtypes A and B are mainly based on G antigen and sequence variations, and G is the most variable gene sequence; these two subtypes can be further distinguished and identified into several genotypes, including 15 different B genotypes and 9 different A genotypes; RSV is constantly evolving globally, and new genotypes have emerged over the years; B genotype diversity is greater than RSV A; RSV F protein is more conserved than G protein in A and B genotypes, and is the main target for vaccine development; F protein differs by only 25 amino acids between A and B genotypes; F protein contains antigenic sites shared by pre- and post-fusion conformations, as well as sites unique to each conformation; six major antigenic sites are defined on the F protein (Ø, I, II, III, IV, V); some antigenic sites are expressed on both Pre-F and Post-F, while others exist only in one conformation; antigenic sites I, II, III, and IV exist in both Pre-F and Post-F conformations and exhibit different affinities; antigenic sites Ø and V only exist in the Pre-F conformation and have the highest affinity for neutralizing antibodies;

[0006] RSV neutralizing activity in human serum mainly comes from antibodies targeting the F protein in the pre-fusion conformation, more specifically, antigenic sites Ø and V; immunological analysis shows that more than 85% of highly effective antibodies are specific to pre-F; in addition, pre-F-specific antibodies are more effective than pre-F and post-F cross-reacting antibodies and also more effective than post-F-specific antibodies; in contrast, no viral neutralizing activity is observed for G protein-specific serum;

[0007] 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 shown that it can protect infants from RSV through maternal vaccination; the monovalent vaccine Arexvy is based on F protein antigen derived from the A2 strain.

[0008] As described above, the pre-F protein is the main target of vaccine development; it is well known that there are still many challenges for RSV vaccine products, and there are many technical problems to be solved, which can be summarized as follows: first, how to better stabilize the trimer antigen, so that the anti-original terminal is stable in the pre-F conformation, and the transition to the post-F conformation is reduced; second, in order to achieve trimerization in the approved product, the vaccine developer introduces a T4 phage sequence foldon at the amino acid terminal end of the F protein, which poses a threat to the safety and effectiveness of the vaccine; third, the sequence contains a p27 fragment, which needs to be cut off by the furin enzyme of the expression host cell, and experiments show that the efficiency of fulin enzyme cutting is not 100%, and the residual p27 affects the purity and immunogenicity of the vaccine; fourth, in order to maintain the stability of the antigen, the prior art is to freeze-dry the vaccine antigen, and mix it with an adjuvant before use, which affects the convenience of use;

[0009] The present application is directed to the challenges in the current RSV vaccine field, and proposes a solution, which can successfully solve the above problems by implementing the embodiments of the present application. The present application provides a new type of RSV liquid dosage form aluminum adjuvant vaccine, which does not contain the p27 sequence and the C-terminal exogenous sequence, and the developed trimer pre-F has excellent structural stability; animal experiments show that the vaccine of the present application has very good titer. SUMMARY

[0010] In view of the above existing problems, the present application is proposed.

[0011] The present application provides a reconstituted respiratory syncytial virus F protein trimer to solve the problems of conformational stability, process defects and dosage form limitations of existing vaccines.

[0012] To solve the above technical problems, the present application provides the following technical solutions:

[0013] In a first aspect, the embodiments of the present application provide a reconstituted respiratory syncytial virus F protein trimer, which comprises,

[0014] a) deleting the p27 fragment SEQ No. 10 between the F2 domain SEQ No. 8 and the F1 domain SEQ No. 9, and connecting the F2 and F1 domains through a GS linker;

[0015] b) connecting each monomer of the trimer through an interchain covalent bond to form a stable pre-F conformation;

[0016] c) the protein retains the neutralizing activity epitope Ø and has immunogenicity.

[0017] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the monomer sequence F2-(GS)n-F1 structure, n≥1, and the F2 domain does not contain a 25-amino acid signal peptide of viral origin.

[0018] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the inter-chain covalent bond is a disulfide bond formed by replacing cysteine, and each monomer has at least one pair of amino acids replaced by cysteine.

[0019] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the cysteine replacement is selected from one of the following five combinations:

[0020] S146C and N460C (SEQ No. 3),

[0021] A149C and Y458C (SEQ No. 4),

[0022] S150C and N460C (SEQ No. 5),

[0023] A153C and Y458C (SEQ No. 6),

[0024] A153C and K461C (SEQ No. 7).

[0025] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the GS linker n value is 1-3, preferably the linker sequence when n=1 is GS (SEQ No. 6-7).

[0026] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the N-terminal signal peptide adopts the CHO cell secretion signal peptide MEWSWVFLFFLSVTTGVHS, realizing the secretion of the trimer protein to the extracellular medium.

[0027] As a preferred solution of the restructured respiratory syncytial virus F protein trimer, in the trimer protein has a purity of ≥95% after purification, and an EC50≤0.20 ng / ml after being stored at 37℃ in a pH 7.6-8.0 buffer for 28 days.

[0028] In a second aspect, the present application provides a vaccine composition comprising the F protein trimer and a pharmaceutically acceptable adjuvant, wherein the aluminum adjuvant content is 0.35 mg / dose.

[0029] As a preferred scheme of the reconstituted respiratory syncytial virus F protein trimer, the trimer protein content is 2.5-10 μg / dose, and the specific antibody GMT is increased by more than 300 times after three immunizations.

[0030] In a third aspect, the application provides use of the reconstituted respiratory syncytial virus F protein trimer in the preparation of a medicament for preventing respiratory syncytial virus infection.

[0031] The application has the beneficial effects that the application designs a RSV F protein trimer, the monomer of the trimer does not contain a p27 sequence, and a furin enzyme cleavage peptide segment aa110~aa136 is deleted; and a GS linker is used for connecting between an F2 domain and an F1 domain.

[0032] The application also designs point mutation combinations, the point mutation combinations can form an inter-chain disulfide bond, so that covalent connection of the trimer is realized, and the problem of trimer depolymerization is fundamentally solved; the trimer is stabilized in a pre-F state, and there is no post-F-like structure, ELISA results show that neutralizing epitopes are completely retained; a method of thermal acceleration experiment is used to challenge the trimer antigen structure, and results show that the trimer antigen purity reaches more than 95% after being placed at 37℃ for 28 days, and the stability is very good; and the trimer is used to prepare a vaccine, and the vaccine can meet the standard of a regulatory requirement without freeze-drying.

[0033] The application deletes a sequence p27 in the F protein, the p27 sequence is SEQ No.10 ELPRFMNYTLNNAKKTNVTLSKKRKRR, preferably, 6 amino acids NNRARR of a furin enzyme cleavage site at an N terminal of the p27 sequence are deleted, 9 hydrophobic amino acids FLGFLLGVG at a C terminal are deleted, and the deleted part is replaced with a GS linker; the GS linker provides a flexible connection for the F2 and F1 peptide segments, and helps the F2 and F1 segments to be combined together and stabilize the pre-F structure; an alpha helix domain downstream of the GS linker is a sequence SEQ No.11 SAIASGVAVSKVL, and the secondary structure is close to a beta sheet structure SEQ No.12 GNTLYYVNKQE of another chain in terms of a crystal structure; in addition, a pair of cysteines is mutated at a selected mutation point on the alpha helix and the beta sheet, so that inter-chain covalent connection is realized.

[0034] The application designs 5 groups of mutation points, which are S146C and N460C, A149C and Y458C, S150C and N460C, A153C and Y458C, and A153C and K461C combinations; one pair of combinations is selected to form a trimer. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0036] Figure 1 Figure is a SWISS-MODEL online pre-F A2 strain modeling diagram of the present application;

[0037] Figure 2 Figure is a pre-F trimer mutant stock SDS-PAGE electrophoresis result diagram of the present application;

[0038] Figure 3 Figure is a pre-F trimer stability HPLC detection result diagram (1-28d-2~8℃) of the present application;

[0039] Figure 4 Figure is a pre-F trimer stability HPLC detection result diagram (1-28d-37℃) of the present application;

[0040] Figure 5 Figure is a pre-F trimer stability HPLC detection result diagram (2-28d-2~8℃) of the present application;

[0041] Figure 6 Figure is a pre-F trimer stability HPLC detection result diagram (2-28d-37℃) of the present application;

[0042] Figure 7 Figure is a pre-F trimer 2~8℃ stability EC50 detection result diagram of the present application;

[0043] Figure 8 Figure is a pre-F trimer 37℃ stability EC50 detection result diagram of the present application;

[0044] Figure 9 Figure is a RSV A vaccine immune mouse GMT comparison analysis result diagram of the present application. DETAILED DESCRIPTION

[0045] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the drawings of the specification.

[0046] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order not to unnecessarily obscure aspects of the present application.

[0047] Secondly, the "one embodiment" or "embodiment" referred to herein means a specific feature, structure, or characteristic that can be included in at least one implementation of the present application. The "in one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is independent or alternative to other embodiments.

[0048] Embodiment 1, which provides the design of F protein monomer amino acid sequence and the construction of expression vector:

[0049] The A2 strain sequence of RSV was obtained from the NCBI database, the A sequence was adjusted (S155C and S290C), and the N-terminal 25 amino acid signal peptide and the C-terminal palmitoylation sequence were deleted, thereby obtaining the starting sequence SEQ No. 1. Further, the p27 and the furin enzyme N-terminal cleavage site amino acids were deleted, a total of 42 amino acids were deleted, including N-terminal NNRARR, and C-terminal FLGFLLGVG was additionally deleted, thereby obtaining SEQ No. 2. The sequence of SEQ No. 2 was introduced into SWISS-MODEL online modeling, and a structure model was obtained after running, which is described in detail in Figure 1 Molecular side view and top view; Figure 1 In the above description, the left side is a side view, and the three monomers can be seen to be close to each other at the C-terminus; the right side is a top view, and the three monomers can be seen to be symmetrically distributed. As can be seen from the figure, the molecule presents a trimer-like structure, which is the pre-F trimer form of RSV F protein. By observing the pre-F spatial structure of RSV, according to the principle of structural biology, in order to stabilize the structure, point mutations are made to the amino acids in the contact part of the three monomers, and covalent bonds are formed on the contact surface. Covalent bonding is superior to hydrophobic bonding or salt bridge bonding, and covalent bonding is not affected by the ionic strength, pH value and buffer system of the solution, and can always keep the relative positions of the single chains correct. This can make the trimer stable throughout the preparation process and have activity, and is also convenient for subsequent industrial process development. Based on SEQ No. 2, five groups of mutants were designed, and the amino acid sequences were SEQ No. 3, SEQ No. 4, SEQ No. 5, SEQ No. 6 and SEQ No. 7, respectively.

[0050] In order to clearly show the design scheme of the mutant, the protein mutant design abstract is listed in Table 1.

[0051]

[0052] According to the above table, Nanjing Kingsway Company was commissioned to conduct codon optimization and whole gene synthesis to construct an expression vector.

[0053] Example 2 provides expression and preliminary cloning screening of recombinant mutants to obtain antigen-expressing strains:

[0054] A CHO K1 cell suspension of 0.8 ml (1.5 x 106 cells / ml) was prepared. Linearized plasmid 20 μg and transfection reagent were added in sequence. The Gene Pulser Xcell (Bio-Rad) was set to a pulse voltage of 300 V, a single pulse of 900 μF, an infinite resistance, and a gap of 4 mm in a disposable shock cup (Bio-Rad). The shock cup was shocked for 12-20 ms. The cells in the shock cup were transferred to a culture flask, and 30 ml of CHO culture solution was added. The flask was cultured at 37°C and 5% CO2 on a shaker at 136 rpm for 24 hr. The cells were collected by low-speed centrifugation at 800 rpm. The cells were transferred to a 96-well flat-bottom culture plate by limiting dilution. The plate was cultured in a 37°C, 5% CO2 incubator. The plate was observed under an inverted microscope, and single clone cell wells were marked. The cells were then screened in 24-well and 6-well cell culture plates by coating with epitope III antibody and detecting the Ø epitope by ELISA. The positive cells were continuously subcultured to establish a cell bank.

[0055] Example 3 provides fermentation and purification of recombinant mutants:

[0056] The cell strains screened in Example 2 were separately subjected to shake flask fermentation culture according to conventional CHO cell fermentation technology. After the fermentation was completed, the cell fermentation broth was first clarified by centrifugation. The clarified broth was subjected to Capto DeVirS gel chromatography to remove most of the host cell proteins and nucleic acids. The broth was then subjected to strong anion exchange chromatography, gel filtration chromatography, and weak anion exchange chromatography for further purification to remove residual host cell proteins and nucleic acids and to remove pyrogens, thereby obtaining high-purity recombinant protein, i.e., a recombinant protein stock solution. The stock solution was stored using the following formula: 20 mM Tris-HCl (containing 150 mM NaCl, 0.5‰ Tween 80). The sample was subjected to SDS-PAGE detection, and the detection results are shown in Figure 2, wherein lane 1, 2, 3, 4, 5 respectively correspond to SEQ No. 3, 4, 5, 6, 7 reduced electrophoresis, lane 7, 8, 9, 10, 11 respectively correspond to SEQ No. 3, 4, 5, 6, 7 non-reduced electrophoresis, the target protein band in the reduced electrophoresis has a molecular weight of 53 KDa, and the non-reduced electrophoresis band has a theoretical molecular weight of 156 KDa, which can be judged as a trimer. According to the results of Figure 2 , it is shown that the pre-F trimer can be obtained in all the five groups of mutant schemes, which meets the design requirements.

[0057] Example 4 provides a pre-F trimer solution stability test:

[0058] In this example, the protein mutant 1 in Example 3 is taken as an example for test description, and the preparation and grouping are carried out according to Table 2 below. The buffer systems and pH values of the experimental groups are different, the ionic strengths are consistent, the pre-F trimer solution is prepared, and is divided into 2ml vials, 1ml / vial. Respectively placed at 2-8℃ and 37℃, and the protein purity is detected by HPLC method every 7 days.

[0059]

[0060] The purity results are shown in Table 3. The purity of experimental group 1 and experimental group 2 is 98.7% and 97.3% respectively at 2-8℃ on the 28th day, and the purity is 97.5% and 98.8% respectively at 37℃ on the 28th day. The detected HPLC spectrum, i.e. the HPLC purity on the 28th day, is shown in Figure 3 , Figure 4 , Figure 5 and Figure 6 , the pre-F maintains the trimer state and does not appear to be degraded.

[0061]

[0062] The results show that the covalently linked RSV pre-F mutant of the application can form a trimer conformation, and is very stable. In different buffer solutions of 20mM PB (pH8.0, containing 135mM NaCl and 0.5‰ Tween-80) and 20mM Tris (pH7.6, containing 135mM NaCl and 0.5‰ Tween-80), under different pH conditions, the trimer state can be maintained, the thermal stability is good, the purity reaches more than 97%, and the purity standard for subsequent preparation of vaccines is completely met.

[0063] Example 5 provides detection of neutralizing activity epitope Ø:

[0064] The samples in experimental group 2 in Example 4 were detected by a double-antibody sandwich method, in which the epitope antibody (1 μg / ml) was coated at 4°C overnight, the liquid in the wells was discarded, 300 μl of PBST was washed 3 times, 250 μl of 2% BSA solution was blocked at 37°C for 1 hour, the liquid in the wells was discarded, 300 μl of PBST was washed 3 times; the pre-F trimer protein to be detected was added, 1 μg / ml, 3-fold gradient dilution, 37°C incubation for 1 hour, the liquid in the wells was discarded, 300 μl of PBST was washed 3 times; the biotin-labeled epitope III antibody (100 ng / ml) was added, 37°C incubation for 1 hour, the liquid in the wells was discarded, 300 μl of PBST was washed 3 times, the Peroxidase-conjugated Streptavidin (20 ng / ml) was added, 37°C incubation for 1 hour, the liquid in the wells was discarded, 300 μl of PBST was washed 5 times, the color developing solution was added, incubation for 15 minutes, the stop solution was added, and the 450 nM absorbance value was read.

[0065] The results are shown in Table 5. Figure 7 and Figure 8 The EC50 of the samples stored at 2-8°C for 7 days, 14 days, 21 days and 28 days was 0.47 ng / ml, 0.47 ng / ml, 0.29 ng / ml and 0.27 ng / ml, respectively; the EC50 of the samples stored at 37°C for 7 days, 14 days, 21 days and 28 days was 0.39 ng / ml, 0.49 ng / ml, 0.46 ng / ml and 0.20 ng / ml, respectively. It can be seen that the neutralizing epitope Ø is stable.

[0066] Example 6 provides a preparation of an aluminum adjuvant vaccine:

[0067] Referring to the dose design of the marketed product, the self-made aluminum adjuvant (0.7 mg / ml) of the company was used for the preparation of the RSV pre-F trimer vaccine, and the specific steps were as follows: the antigen stock solution was diluted with dilution buffer to 240 μg / ml, and the diluted stock solution was slowly added into an equal volume of aluminum adjuvant suspension while mixing. Then, 0.6 ml / branch was divided into 2 ml sinter bottles, and the prepared vaccine antigen content was 60 μg per dose, which was a RSV type A monovalent vaccine.

[0068] Example 7 RSV A type vaccine immunogenicity research

[0069] According to the vaccine preparation method in Example 6, the vaccine for animal experiments was prepared, BALB / c mice were repeatedly immunized, and the immunogenicity of the vaccine was evaluated. The specific grouping is shown in Table 4.

[0070]

[0071] The animals were randomly divided into 7 groups, 10 animals per group, and immunized by intraperitoneal injection, with an immunization dose of 0.5 ml / animal. Immunization: 21 days and 42 days after the first immunization, the animals were immunized again, for a total of 3 times. Blood collection: tail vein blood samples were collected from the mice after the first and second immunizations, and the mice were enucleated for blood collection after the third immunization. Blood collection time points: a total of 3 blood collections were performed, on days 21, 42 and 63 after the first immunization. The blood samples were placed in 1.5 ml centrifuge tubes, and left to stand at room temperature for 2 hours, then transferred to 2-8°C overnight, centrifuged at 5000 rpm for 5 minutes, and the serum was separated and stored at -20°C or below.

[0072] ELISA method for detecting antibody titers: RSV type A antigen was coated on a blank enzyme-labeled plate, and a series of pre-diluted mouse serum samples were added, and reacted at 37°C for 60 minutes; goat anti-mouse IgG-HRP conjugate was added, and reacted at 37°C for 60 minutes; TMB color developing solution was added for color development, and stop solution was added to stop the reaction; the absorbance value was measured by an enzyme-labeled instrument (450 nm). The cutoff value was 3 times the average absorbance value of the negative control (aluminum hydroxide adjuvant mouse serum) (if the absorbance value is less than 0.1, it is calculated as 0.1), and the maximum dilution corresponding to the average absorbance value of the immune mouse serum that is higher than the cutoff value is the antibody titer of the immune mouse serum.

[0073] The results are shown in Table 5 below. In the aluminum adjuvant-containing group, the GMT D63 was 294, 313 and 325 times higher than the GMT D21 after immunization with different doses of antigen, respectively. In the group without adjuvant, the GMT D63 was 73, 134 and 61 times higher than the GMT D21 after immunization with different doses of antigen, respectively. Thus, the pre-F trimer has good immunogenicity as a vaccine antigen, and aluminum adjuvant can significantly increase the GMT level, and the data support the use of the pre-F trimer protein antigen as an RSV type A vaccine.

[0074]

[0075] The data in Table 5 were analyzed, as shown in Figure 9 From the analysis of the dose, adjuvant and immunization durability, it can be seen that the pre-F trimer antigen prepared by the present application is stable and has good immunogenicity, and can be used to prepare a vaccine for the prevention of RSV type A virus disease.

[0076] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A reconstructed respiratory syncytial virus F protein trimer, characterized in that: The monomeric amino acid sequence is selected from SEQ No. 3, SEQ No. 4, SEQ No. 5, SEQ No. 6 or SEQ No.

7. The trimer is formed by three monomers covalently linked by interchain disulfide bonds to form a stable pre-F conformation before fusion.

2. The reconstructed respiratory syncytial virus F protein trimer as described in claim 1, characterized in that, After purification, the purity is ≥95%, and after storage at 37°C for 28 days in pH 7.6-8.0 buffer, the EC50 is ≤0.20 ng / ml.

3. A vaccine composition based on a reconstructed respiratory syncytial virus F protein trimer according to any one of claims 1-2, characterized in that, It includes the F protein trimer and a pharmaceutically acceptable adjuvant, wherein the aluminum adjuvant content is 0.35 mg / dose.

4. The vaccine composition according to claim 3, characterized in that, The F protein trimer content is 2.5-10 μg / dose.

5. Use of a reconstructed respiratory syncytial virus F protein trimer as described in any one of claims 1-2 in the preparation of a medicament for the prevention of respiratory syncytial virus infection.

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