A rsv vaccine composition comprising rsv pre-f protein, and methods of making and using the same

Abstract

The present application relates to the technical field of biological vaccine, in particular to a RSV vaccine composition containing RSV pre-F protein, a preparation method and application thereof. The vaccine composition contains RSV pre-F protein and adjuvant; wherein the residual proportion of pep27 fragment in the RSV pre-F protein is not higher than 9.08%. The present application also provides a preparation method of the vaccine composition and application thereof in preparing a medicine for preventing respiratory syncytial virus infection. The present application improves the immunogenicity of RSV pre-F antigen by controlling the residual proportion of pep27 fragment and optimizing the combination of adjuvant, can induce higher level of humoral immune and cellular immune response, and has good challenge protection potential and safety.

Description

Technical Field

[0001] This invention relates to the field of biological vaccine technology, specifically to an RSV vaccine composition containing RSV pre-F protein, its preparation method, and its application. Background Technology

[0002] Respiratory syncytial virus (RSV) is a major pathogen causing acute lower respiratory tract infections in infants, the elderly, and immunocompromised individuals, leading to severe respiratory diseases such as bronchiolitis and pneumonia, and posing a significant disease burden. RSV surface fusion protein F plays a crucial role in viral invasion of host cells and is a primary target antigen for inducing neutralizing antibodies; therefore, vaccine development focusing on the RSV F protein has become an important technological direction in this field.

[0003] In existing technologies, RSV vaccine antigen design primarily focuses on the F protein, especially the pre-fusion conformation of the F protein (pre-F). The RSV F protein is synthesized in its precursor form and cleaved by host proteases to form the F2, pep27, and F1 fragments. The mature F protein does not contain the pep27 fragment. Furthermore, the pre-F conformation can present more key epitopes associated with highly efficient neutralizing activity, and pre-F-specific antibodies play a crucial role in determining RSV neutralizing activity. As RSV vaccine research continues to advance, vaccines based on the stabilized pre-F protein have become a significant technological approach.

[0004] However, existing technologies still have shortcomings. On the one hand, if the precursor of RSV pre-F antigen is not fully cleaved during preparation, it can easily lead to residual PEP27 fragments, thereby increasing the proportion of immature antigens and affecting the proportion of effective mature pre-F antigens and the exposure of key neutralizing epitopes, which is detrimental to obtaining ideal immunogenicity. On the other hand, even when using pre-F antigens, different types and amounts of adjuvants have significantly different effects on enhancing humoral and cellular immunity. Existing technologies still do not adequately consider the synergistic effect of antigen maturity control and adjuvant compatibility optimization. Therefore, developing an RSV vaccine composition that can control the residual proportion of PEP27 fragments in RSV pre-F antigens, enhance humoral and cellular immune responses through reasonable adjuvant combinations, and balance challenge protection efficacy and safety remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides an RSV vaccine composition containing RSV pre-F protein, its preparation method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An RSV vaccine composition containing RSV pre-F protein, the vaccine composition comprising RSV pre-F protein and an adjuvant; The residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 9.08%.

[0007] Preferably, the residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 5.20%.

[0008] Preferably, the residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 1.87%.

[0009] Preferably, the RSV pre-F protein does not contain the pep27 fragment.

[0010] Preferably, the adjuvant is selected from at least one of MF59, aluminum adjuvant, and CpG adjuvant.

[0011] Preferably, the adjuvant is MF59.

[0012] Preferably, the vaccine composition contains 12-48 μg of RSV pre-F protein per dose and 12-45 μL of MF59 per dose.

[0013] Preferably, the vaccine composition contains 12 μg of RSV pre-F protein per dose and 25 μL of MF59 per dose.

[0014] A method for preparing the RSV vaccine composition includes the step of mixing RSV pre-F protein with an adjuvant, wherein the residual proportion of the pep27 fragment in the RSV pre-F protein is not higher than 9.08%.

[0015] The use of the RSV vaccine composition or the vaccine composition prepared by the method in the preparation of a medicament for the prevention of respiratory syncytial virus infection.

[0016] This invention addresses the shortcomings of existing RSV vaccine compositions in terms of immunogenicity, formulation optimization, and safety. It provides an RSV vaccine composition containing RSV pre-F protein and its preparation method. This vaccine composition uses RSV pre-F protein as the antigen and is used in conjunction with an adjuvant. By controlling the residual proportion of the pep27 fragment in the RSV pre-F protein to no more than 9.08%, antigen maturity and immunogenicity are improved. When the residual proportion of the pep27 fragment is at a low level, the antigen maintains good neutralizing antibody induction ability; conversely, when the residual proportion of the pep27 fragment increases, the level of neutralizing antibodies induced by the antigen decreases significantly. Therefore, the residual proportion of the pep27 fragment can serve as an important quality control indicator for RSV vaccine antigens.

[0017] The beneficial effects of this invention are: 1. This invention provides an RSV vaccine composition containing RSV pre-F protein, its preparation method, and its application. This invention is the first to define the residual proportion of the pep27 fragment as one of the important quality control parameters for RSV pre-F antigen. When the residual proportion of the pep27 fragment is within the range of 0-9.08%, the antigen-induced neutralizing antibody titer remains at a relatively high level; however, when the residual proportion of the pep27 fragment increases to 18.82%, the neutralizing antibody titer decreases significantly. Controlling the residual proportion of the pep27 fragment helps to reduce the proportion of immature precursor antigen and increase the proportion of effectively mature pre-F antigen, thereby promoting the presentation of key neutralizing epitopes and enhancing antigen immunogenicity.

[0018] 2. Through screening different types and dosages of adjuvants, MF59, aluminum hydroxide, or CpG1018 were found to enhance the immune response induced by RSV pre-F antigen to a certain extent, with MF59 showing a superior enhancing effect. When the RSV pre-F antigen content was 12 μg / 0.05 mL and the MF59 content was 25 μL / 0.05 mL, the vaccine composition could induce high levels of specific IgG antibodies, neutralizing antibodies, and IFN-γ secretion. The vaccine composition of this invention combines the advantages of enhancing both humoral and cellular immunity.

[0019] 3. The preferred vaccine composition of the present invention can induce high levels of specific binding antibodies and neutralizing antibodies, and exhibits good immunogenicity and anti-RSV efficacy in a cotton rat model. At the same time, compared with the formalin-inactivated vaccine FI-RSV, the preferred vaccine composition of the present invention does not show obvious enhancement of lung pathological damage, and has good safety while exerting immune protection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. 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.

[0021] Figure 1 This is a schematic diagram showing the RSV-F antigen-specific IgG antibody titers in the serum of mice after immunization in each group.

[0022] Figure 2 This is a schematic diagram showing the NT50 titer of neutralizing antibodies against RSV A2 strain in the serum of mice after immunization in each group.

[0023] Figure 3 This is a schematic diagram showing the NT50 titer of neutralizing antibodies against RSV B strain in the serum of mice after immunization in each group.

[0024] Figure 4 This diagram illustrates the level of IFN-γ secreted by spleen cells in mice after immunization in each group.

[0025] Figure 5 This is a diagram illustrating the weight changes of cotton mice.

[0026] Figure 6 This is a schematic diagram showing the titer of specific binding antibodies in the serum of cotton rats.

[0027] Figure 7 This is a schematic diagram of the NT50 titer of neutralizing antibodies in the serum of cotton rats.

[0028] Figure 8 This is a schematic diagram of the pathological scoring (total HE staining score) of cotton rat lung tissue. Detailed Implementation

[0029] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0030] 1. Antigen structure description 1.1 Structural characteristics of RSV pre-F antigen RSV F protein exists in precursor form. The RSV F protein precursor is cleaved by furin protease to form F2, pep27, and F1 fragments, with the specific amino acid sequences as follows. The pep27 fragment is a cleavage fragment of the precursor protein; the mature RSV F protein does not contain the pep27 fragment. The RSV F protein peptide contains F1 and F2 fragments, which are linked by disulfide bonds to form basic structural units called protomers. Multiple protomers further assemble to form the mature RSV F protein trimer.

[0031] The amino acid sequence of the pep27 fragment is as follows: ELPRFMNYTL NNAKKTNVTL SKKRKRR.

[0032] The amino acid sequence of the F1 fragment is as follows: FLGFLLGVGS AIASGVAVCK VLHLEGEVNKIKSALLSTNK AVVSLSNGVS VLTFKVLDLK NYIDKQLLPI LNKQSCSISN IETVIEFQQK NNRLLEITREFSVNAGVTTP VSTYMLTNSE LLSLINDMPI TNDQKKLMSN NVQIVRQQSY SIMCIIKEEV LAYVVQLPLYGVIDTPCWKL HTSPLCTTNT KEGSNICLTR TDRGWYCDNA GSVSFFPQAE TCKVQSNRVF CDTMNSLTLPSEVNLCNVDI FNPKYDCKIM TSKTDVSSSV ITSLGAIVSC YGKTKCTASN KNRGIIKTFS NGCDYVSNKGVDTVSVGNTL YYVNKQEGKS LYVKGEPIIN FYDPLVFPSD EFDASISQVN EKINQSLAFI RKSDELLSAIGGYIPEAPRD GQAYVRKDGE WVLLSTFL.

[0033] The amino acid sequence of the F2 fragment is as follows: QNITEEFYQS TCSAVSKGYL SALRTGWYTSVITIELSNIK ENKCNGTDAK VKLIKQELDK YKNAVTELQL LMQSTPATNN RARR.

[0034] RSV F protein peptide is the mature RSV F protein trimer. In nature, the mature RSV F protein trimer mediates the fusion of the virus and the cell membrane. The pre-fusion conformation of the mature RSV F protein trimer (called "pre-F") is highly unstable. Once the RSV virus docks with the cell membrane, the RSV F protein trimer undergoes a series of conformational changes and transitions to a highly stable post-fusion conformation (called "post-F").

[0035] The antigen involved in this invention is the RSV pre-F antigen. Compared with other conformational states, the pre-F conformation can more effectively present key epitopes related to the induction of neutralizing antibodies, and therefore serves as an immunogen for RSV vaccines.

[0036] The Pre-F structure of the protein was confirmed using ELISA. The Pre-F protein belongs to the pre-fusion conformation, not the post-fusion conformation. Epitope Site II and its corresponding antibody MOTA are present in both the pre-fusion and post-fusion structures, while epitope Site Ø is a pre-fusion specific epitope, with corresponding antibody D25. Additionally, antibody AM14 specifically binds to the pre-fusion trimer epitope, as shown in the table below:

[0037] The binding ability of the pre-F protein and the commercially available post-fusion protein to MOTA antibody, D25 antibody, and AM14 antibody was tested to confirm that it belongs to the pre-F structure.

[0038] MOTA: 2 μg / ml MOTA antibody was coated onto a microplate, protein was added in a gradient, and then HRP-GoatAnti-Human-IgG was added for colorimetric reaction. Four-parameter fitting showed the expected results, i.e., all exhibited typical "S"-shaped curves. D25: 2 μg / ml MOTA antibody was coated onto a microplate, pre-F protein and commercially available post-F protein were added in a gradient, and then HRP-modified D25 antibody was added for colorimetric reaction. Four-parameter fitting showed the expected results, i.e., only pre-F protein showed a typical "S"-shaped curve, and commercially available post-F protein did not specifically bind to D25. AM14: 2 μg / ml MOTA antibody was coated onto a microplate, pre-F protein and commercially available post-F protein were added in a gradient, and then HRP-modified AM14 antibody was added for colorimetric reaction. Four-parameter fitting showed the expected results, i.e., only pre-F showed a typical "S"-shaped curve, and commercially available post-F protein did not specifically bind to AM14. This confirms it as a Pre-F structure, as shown in the table below:

[0039] 1.2 Relationship between the residual proportion of PEP27 and the degree of antigen maturation Since the pep27 fragment is a cleavage fragment during the processing of RSV F protein precursors, the residual proportion of the pep27 fragment can be used to reflect the degree of antigen maturation. Generally speaking, a higher residual proportion of the pep27 fragment indicates a higher proportion of insufficiently cleaved precursor antigen, which may reduce the proportion of effectively matured pre-F antigen and further affect the effective presentation of neutralizing epitopes.

[0040] 2. Experimental Example 1: The effect of different pep27 residual ratios on the immunogenicity of RSV pre-F antigen The purity of RSV pre-F was determined using reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC separates different components in the sample based on the differences in hydrophobic interactions between the sample and the hydrophobic groups of the stationary phase. Since there is a difference in hydrophobicity between pep27-containing proteins and normal proteins, the proportion of pep27-containing proteins can be characterized. After separation using a Waters XBridge R ProteinBEH C4 300Å 3.5μm, 4.6mm*150mm column, the signal was acquired at 214nm for purity analysis. The acquisition equipment was a Water e2695 HPLC system, and the HPLC conditions were: mobile phase A was 0.1% TFA water, mobile phase B was 0.1% TFA acetonitrile, gradient elution was performed at a flow rate of 0.5ml / min, and the elution gradient varied from 2%B to 70%B within the range of 0.1min–25min. The data analysis software was Empower v3.81, and the purity was calculated using the area normalization method.

[0041] RSV pre-F antigens with different residual proportions of the pep27 fragment were selected and vaccine compositions were prepared under the same antigen dosage and adjuvant conditions. In the vaccine compositions, the Pre-F antigen dosage was 12 μg and the MF59 adjuvant dosage was 25 μL. The immunogenicity of samples with different residual proportions of the pep27 fragment was compared using the neutralizing antibody titer (NT50, geometric mean titer, GMT) against RSV A2 and RSV B strains in the serum of immunized animals as the evaluation index, as shown in Table 1.

[0042] Table 1. RSV pre-F antigen-induced neutralizing antibody titers based on the residual proportion of the pep27 fragment

[0043] This example investigated the relationship between the retention rate of the pep27 fragment in RSV F protein and the neutralizing antibody titer (NT50 geometric mean titer, GMT). The results are shown in Table 1. When the residual pep27 fragment rate was in the range of 0-9.08%, the antigen-induced neutralizing antibody titer values ​​did not differ significantly; however, when the residual pep27 fragment rate was as high as 18.82%, the neutralizing antibody titer value decreased significantly.

[0044] The above results indicate that an excessively high proportion of residual PEP27 fragments reduces the immunogenicity of RSV pre-F antigens. This may be because an increased proportion of residual PEP27 fragments implies a higher proportion of immature antigens, thereby reducing the proportion of effectively mature pre-F antigens and affecting exposure to key neutralizing epitopes. Therefore, controlling the residual PEP27 fragment proportion is crucial for obtaining highly effective pre-F vaccines. In conclusion, the PEP27 proportion can serve as a key quality control indicator for RSV vaccines, and minimizing its residual levels is an important strategy for developing highly immunogenic RSV vaccines.

[0045] 3. Experimental Example 2: Effects of different adjuvants and dosages on immune response This study aimed to investigate the effects of different adjuvant types and dosages on the level of immune response to RSV pre-F antigen, and to screen for vaccine composition formulations that could form a better compatibility with RSV pre-F antigen. Evaluation indicators included humoral immunity and cellular immunity. Humoral immunity was evaluated using specific IgG antibody titers and neutralizing antibody titers, while cellular immunity was evaluated using the ability of spleen cells to secrete IFN-γ.

[0046] 3.1 Preparation of vaccine composition To compare the effects of different antigen doses, adjuvant types, and adjuvant dosages on the immune response, this study set up 9 experimental groups. The single-dose administration system for each vaccine composition was calculated in 50 μL / dose volumes. The component dosages are shown in Table 2. Each 50 μL system was prepared, made up with PBS, gently mixed by pipetting, stored in the dark, and administered to experimental animals within 2 hours.

[0047] Table 2. Dosage of each component in the vaccine composition

[0048] 3.2 Animal immunization and sample collection Immunization experiments were conducted using BALB / c mice. Animals in each group were immunized twice, on day 0 and day 21, with the same dose of the vaccine composition. To ensure comparability of results across groups, a consistent dosing schedule and volume were used for all groups.

[0049] Serum samples were collected from mice on Day 21 and Day 42 after immunization. 100 μL of blood was collected from each mouse. The serum was separated and aliquoted into 50 μL vials, with at least 2 vials per sample. The vials were stored at -80℃ and used to determine the level of IgG antibodies in the serum by ELISA. The level of neutralizing antibodies in the serum was detected by immunofluorescence spot assay.

[0050] On Day 42, mice were sacrificed, spleen cells were isolated, cultured overnight, and used to determine the level of specific T cell immune response using the ELISpot method.

[0051] 4. Immunogenicity testing 4.1 Humoral immunity assessment (ELISA method) Serum from mice on Day 21 and Day 42 post-immunization was collected for ELISA testing. The specific procedure is as follows: Coating: Dilute the antigen protein with alkaline coating buffer and add it to the ELISA plate, incubate overnight at 4°C.

[0052] Blocking: On the second day, discard the coating solution, wash the microplate with PBST and pat dry, then add blocking solution and incubate at 37°C for 1 hour.

[0053] Serum binding: Discard the blocking solution, wash the ELISA plate with PBST, pat dry, and label the plate with group numbers. Add the diluted serum to the corresponding wells and incubate at 37°C for 1 hour.

[0054] Secondary antibody incubation: Discard the serum diluent, wash the ELISA plate with PBST and pat dry, add the diluted secondary antibody, and incubate at 37°C for 1 hour.

[0055] Color development: Discard the secondary antibody, wash the microplate with PBST and pat dry, add the color development solution, and let it stand at room temperature in the dark for 5-10 minutes. When the color of the well plate shows a clear gradient, add the stop solution to terminate the reaction.

[0056] Reading: OD450 value is measured by microplate reader within 15 minutes after adding stop solution.

[0057] Data processing: The cutoff was 2.1 times the negative OD value, and the maximum serum dilution with an OD value greater than the cutoff was used as the antibody titer.

[0058] 4.2 Evaluation of humoral immunity (immunofluorescence spot method) Neutralizing antibody tests were performed on serum samples from mice on Day 21 and Day 42 post-immunization, as follows: Preparation of Hep-2 cells: After complete digestion, Hep-2 cells were centrifuged and resuspended, then cultured at 3 × 10⁻⁶ cells per cell. 4 / wells were seeded into 96-well cell plates at 100 μL / well, so that the cell confluence reached more than 90% on the second day.

[0059] Sample processing: Diluted mouse serum was diluted 1:50 with low serum medium in a 96-well plate, and then serially diluted 3-fold, for a total of 8 gradients, with 2 replicates for each gradient. The WVB virus solution with the measured titer was diluted 100-fold with low serum medium to obtain the virus dilution.

[0060] Incubation: Mix the virus diluent and serum diluent at a 1:1 ratio and incubate at 37°C and 5% CO2 for 1 hour. The cell control wells (CC) contain low-serum culture medium, and the virus control wells (VC) contain a mixture of low-serum culture medium and an equal volume of virus diluent.

[0061] Infection: After incubation, add 100 μL of virus-serum mixture to cells at a rate of 2 h for infection.

[0062] Culture: After infection, discard the mixture, add 100 μL of low serum culture medium to each well, and incubate in a carbon dioxide incubator at 37°C and 5% CO2 concentration for 20 h.

[0063] Fixation: Discard the culture medium, add 200 μL of 4% PFA fixative to each well, and wipe the inside and outside of the cap, as well as the bottom and sides of the plate with alcohol or sodium hypochlorite. Open the cap and irradiate with ultraviolet light for 1 hour.

[0064] Membrane disruption: After 1 hour of UV exposure, discard the fixative, wash twice with PBS, gently pat dry, add 100 μL of disruption solution to the cell plate and incubate at room temperature for 0.5 hours, discard the solution, wash twice, and gently pat dry.

[0065] Blocking: 10% FBS+PBS, 200μL / well, incubate at room temperature for 0.5h, discard the solution, wash twice with PBS, and gently pat dry.

[0066] Antibody incubation: Add diluted D25 antibody at a rate of 100 μL / well and incubate at room temperature for 1 h. Discard the solution, wash 5 times with PBS, and gently pat dry.

[0067] Secondary antibody incubation: Add 100 μL of diluted goat anti-human IgG-AF488 to each well, incubate at room temperature in the dark for 1 hour, discard the solution, wash 5 times with PBS, and gently pat dry.

[0068] Counting: Use an enzyme-linked immunosorbent assay (ELISA) speckle analyzer to take pictures and count the samples. The counting should be done on the day the samples are collected. Calculate the neutralizing antibody titer based on the inhibition of viral infection by each serum sample.

[0069] 4.3 Preparation of mouse spleen cell suspension Mice were sacrificed 42 days after immunization, and their spleens were isolated under aseptic conditions for ELISpot assay. The specific procedures are as follows: Grinding: The spleen was ground and filtered in a cell sieve, and the spleen cell suspension was collected and centrifuged at 300×g at room temperature for 5min.

[0070] Lysing red blood cells: Discard the supernatant, add red blood cell lysis buffer to resuspend the precipitate, treat at 37℃ for 5 min, and centrifuge at 300×g for 5 min. Wash with serum-free medium, and finally resuspend the cells in 3 mL of complete medium; the complete medium is prepared by mixing 90% RPMI 1640 medium, 10% FBS and 1% penicillin-dextrin antibiotics.

[0071] Counting: After diluting 10 times for counting and determining cell viability, the cells were cultured overnight in a 37°C, 5% CO2 cell incubator for subsequent enzyme-linked immunospot assay (ELISpot assay) to detect the immune response.

[0072] 4.4 Cellular Immunoassay (ELISpot Method) The above-mentioned mouse spleen cell suspension, cultured overnight, was used for ELISpot detection. The specific procedure is as follows: Incubation: According to the plate layout, add peptide stimulation mixture, non-stimulating control or positive stimulant respectively. Dilute the isolated mouse spleen lymphocytes according to the counting results. Two parallel controls for each sample. Incubate the cells in a 37°C, 5% CO2 cell culture incubator for 36 h. Do not move the ELISpot plate during this period.

[0073] Secondary antibody binding: Add specific detection antibody (secondary antibody), incubate at room temperature for 2 hours, discard the liquid in the plate, and wash 5 times with PBS.

[0074] Enzyme conjugation: Add the diluted enzyme conjugate and incubate at room temperature for 1 hour.

[0075] Color development: After the spots have formed, rinse thoroughly with plenty of distilled water to stop the reaction.

[0076] Spot counting: After the ELISpot plate has dried, spot counting analysis was performed using the ELISpot enzyme-linked spot image analysis system.

[0077] 5. Results 5.1 ELISA method for detecting antibody titers from Figure 1It can be seen that: (1) Compared with the PBS control group (Group 1), the levels of F protein-specific IgG antibodies produced in other groups were higher than those in the negative control group. (2) Among the adjuvant-free antigen groups (Groups 2 to 4), the antibody titer was highest when the pre-F antigen content was 12 μg. (3) Compared with the negative control group and the adjuvant-free antigen group, the IgG antibody levels in each adjuvant group were further increased, indicating that the antibody levels using MF59 adjuvant (Groups 5 to 7) were higher than those using aluminum hydroxide (Group 8) and CpG1018 (Group 9). Among them, when MF59 was 25 μL (Group 6), it could induce higher IgG antibody levels in mice.

[0078] 5.2 Detection of neutralizing antibody titers using immunofluorescence spot assay The levels of neutralizing antibodies against RSV A2 and RSV B viruses in mouse serum were detected 3 weeks (Day 42) after the second immunization using an immunofluorescence dot assay. Figure 2 and Figure 3 The results showed that: (1) Compared with the PBS control group (Group 1) and the adjuvant-free antigen group (Groups 2 to 4), each adjuvant group could increase the level of neutralizing antibodies in mouse serum to varying degrees, and the neutralizing antibody titers against RSV A2 and RSV B viruses in the serum of mice after immunization were significantly higher than those in the negative control group. (2) The neutralizing antibody levels in the MF59 group (Groups 5 to 7) were generally higher than those in the aluminum hydroxide group (Group 8) and CpG1018 (Group 9), and the highest level of neutralizing antibodies could be induced in mice when the dosage of MF59 was 25 μL in Group 6.

[0079] 5.3 ELISpot assay to detect the level of IFN-γ secreted by spleen cells in immunized mice The level of IFN-γ secreted by spleen cells in mice was detected 3 weeks (Day 42) after the second immunization using the ELISpot method. Figure 4 The results showed that: (1) Compared with the PBS control group (Group 1), all groups could stimulate mouse spleen cells to secrete cytokine IFN-γ. Among them, the IFN-γ level induced in the unadjuvanted antigen group (Groups 2 to 4) was relatively low, indicating that although the antigen alone could induce a certain immune response, its effect on enhancing cell immunity was limited. (2) Compared with the PBS control group and the unadjuvanted antigen group, when the adjuvants were MF59, CpG1018 and aluminum hydroxide, the prepared recombinant respiratory syncytial virus vaccine composition could increase the IFN-γ secretion level to varying degrees. Among them, the group using MF59 adjuvant alone had a better immune effect, and when the MF59 content was 25 μL (Group 6), the IFN-γ level secreted by mouse spleen cells was the highest.

[0080] The results of specific IgG antibody titers, neutralizing antibody titers, and IFN-γ assays showed that different adjuvant types and dosages significantly affected the immune response induced by RSV pre-F antigen. Increasing the Pre-F antigen dose alone did not achieve a superior overall immune effect compared to the optimal adjuvant combination; in contrast, combining Pre-F antigen with adjuvants more effectively enhanced humoral and cellular immunity. Furthermore, a Pre-F antigen concentration of 12 μg / 0.05 mL and an MF59 concentration of 25 μL / 0.05 mL induced higher levels of humoral and cellular immune responses in mice.

[0081] 6. Experimental Example 3: Study on the protection against RSV vaccine in a rat model. This study aimed to evaluate the immunogenicity, protective efficacy against RSV infection, and safety of a preferred vaccine composition using a rat model of RSV infection. Specifically, the titers of specific binding antibodies and neutralizing antibodies in the serum of immunized rats were measured, and the results were combined with pathological observations of lung tissue to comprehensively assess the protective effect of the tested vaccine against RSV infection and whether there was any enhancement of lung pathological damage.

[0082] 6.1 Test Groups and Test Substances

[0083] 6.2 Effect of the tested vaccine on the body weight of cotton rats During the trial, from day 0 to 48, weight was measured twice a week; from day 49 to 53, weight was measured daily, and health and survival status were recorded. Results are shown below. Figure 5 .

[0084] The results showed that the weight of all groups of cotton rats remained stable after inoculation with the test substance and remained relatively stable after inoculation with the virus, with no obvious abnormalities observed. These results indicate that the test vaccine was well tolerated in cotton rats.

[0085] 6.3 Effect of the tested vaccine on the titer of specific binding antibodies in rat serum The titer of specific binding antibodies against RSV pre-F protein in rat serum samples on day 35 was detected by ELISA. Results are shown below. Figure 6 The results showed that the antibody titer in the saline group was below the detection limit, and that the FI-RSV inactivated vaccine and RSV A2 / B pre-infection could induce a certain level of antibodies specifically binding to RSV A and RSV B pre-F proteins.

[0086] Furthermore, the geometric mean titer of specific binding antibodies against RSVA pre-F protein in the serum samples of rats in the CHO cell group was 4,166,489 ( ). P <0.0001), the geometric mean titer of the antibody specifically binding to RSV B pre-F protein was 4026668 ( P <0.0001), the geometric mean titer of specific binding antibody against RSV A pre-F protein in the serum samples of rats in the recombinant respiratory syncytial virus vaccine (CHO cells) (adjuvant-free) group was 5257 ( P <0.0001), the specific binding antibody titer against RSV B pre-F protein was 730. These results indicate that the recombinant respiratory syncytial virus vaccine (CHO cells) can induce higher levels of specific binding antibodies in the cotton rat model, showing good immunogenicity.

[0087] 6.4 Effect of the tested vaccine on the titer of neutralizing antibodies in the serum of rats The titers of neutralizing antibodies against RSV A2 and RSV B strains in rat serum samples on day 35 were determined using the micro-neutralization method. The results are shown in [Figure number missing]. Figure 7 The results showed that the neutralizing antibody titers in the saline group were all below the detection limit. FI-RSV induced low levels of RSV A2 and RSV B neutralizing antibodies, and RSV A2 / B pre-infection induced a certain level of neutralizing antibodies. The geometric mean of neutralizing antibody titers against RSV A2 and RSV B9320 in the serum samples of rats in the test substance recombinant respiratory syncytial virus vaccine (CHO cell) group was 9352 ( P <0.0001), 4991 ( P <0.0001), the geometric mean neutralizing antibody titers against RSV A2 and RSV B9320 in the serum samples of cotton rats in the recombinant respiratory syncytial virus vaccine (CHO cells) (without adjuvant) group were 71 and 40, respectively. This result indicates that the tested recombinant respiratory syncytial virus vaccine (CHO cells) exhibited a good neutralizing antibody induction effect.

[0088] 6.5 Effect of the tested vaccine on the degree of lung tissue lesions in a rat infection model HE staining was used to assess lung tissue lesions in experimental animals, mainly examining perivascular infiltration, peribronchiolar infiltration, alveolar infiltration, and interstitial infiltration indices. Results were as follows: Figure 8 The results showed that, compared with the saline group, the formalin-inactivated vaccine FI-RSV significantly enhanced lung pathological damage, while no enhanced lung pathological damage was observed in the recombinant respiratory syncytial virus vaccine (CHO cell) group and the recombinant respiratory syncytial virus vaccine (CHO cell) (without adjuvant) group.

[0089] 7. Conclusion Based on the combined results of changes in body weight, specific binding antibody titers, neutralizing antibody titers, and lung tissue pathological evaluation, the recombinant respiratory syncytial virus vaccine (CHO cells) exhibits good immunogenicity and anti-RSV efficacy in a rat model. This vaccine can induce certain levels of specific binding antibodies and neutralizing antibodies. Furthermore, HE staining assessment of lung tissue lesions in rats showed that, compared to the saline group, the formalin-inactivated vaccine FI-RSV significantly enhanced lung pathological damage, while the recombinant respiratory syncytial virus vaccine (CHO cells) group did not exhibit enhanced lung pathological damage. This demonstrates that the preferred vaccine composition of this invention not only possesses good humoral immune induction ability but also exhibits good challenge protection potential and safety.

Claims

1. An RSV vaccine composition containing RSV pre-F protein, characterized in that, The vaccine composition comprises RSVpre-F protein and an adjuvant; The residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 9.08%.

2. The RSV vaccine composition according to claim 1, characterized in that, The residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 5.20%.

3. The RSV vaccine composition according to claim 1, characterized in that, The residual proportion of the pep27 fragment in the RSV pre-F protein is no higher than 1.87%.

4. The RSV vaccine composition according to claim 1, characterized in that, The RSV pre-F protein does not contain the pep27 fragment.

5. The RSV vaccine composition according to any one of claims 1-4, characterized in that, The adjuvant is selected from at least one of MF59, aluminum adjuvant, and CpG adjuvant.

6. The RSV vaccine composition according to claim 5, characterized in that, The adjuvant is MF59.

7. The RSV vaccine composition according to claim 6, characterized in that, The vaccine composition contains 12-48 μg of RSV pre-F protein per dose and 12-45 μL of MF59 per dose.

8. The RSV vaccine composition according to claim 7, characterized in that, The vaccine composition contains 12 μg of RSV pre-F protein per dose and 25 μL of MF59 per dose.

9. A method for preparing the RSV vaccine composition according to any one of claims 1-8, characterized in that, The step includes mixing RSVpre-F protein with an adjuvant, wherein the residual proportion of the pep27 fragment in the RSV pre-F protein is not higher than 9.08%.

10. The use of the RSV vaccine composition according to any one of claims 1-8 or the vaccine composition prepared by the method of claim 9 in the preparation of a medicament for the prevention of respiratory syncytial virus infection.

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