Recombinant respiratory syncytial virus vaccine and preparation method thereof

Abstract

The invention belongs to the field of vaccines, and provides an adjuvant composition containing MF59 and CpG. The invention discloses a full-liquid preparation RSV vaccine containing the adjuvant composition. The vaccine contains RSV pre-F protein. The preparation method of the all-liquid preparation RSV vaccine comprises the operation of carrying out compatibility on the RSV pre-F protein and the adjuvant composition. According to the adjuvant composition and the full-liquid preparation RSV vaccine based on the adjuvant composition, the conformation of pre-F protein before fusion can be kept stable in a liquid dosage form, the preparation cost of the vaccine can be greatly reduced, and meanwhile the cellular immune effect and the humoral immune effect of the vaccine are improved.

Description

Technical Field

[0001] The invention belongs to the field of vaccines, and particularly relates to a recombinant respiratory syncytial virus vaccine and a preparation method thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the main infectious factors that cause severe respiratory diseases in infants, young children and the elderly. Since the discovery of RSV in 1956, people have launched multiple vaccine development programs. Currently licensed RSV vaccines all use F protein as an immunogen. F protein exists in two main conformational states: pre-fusion conformation (pre-F) and post-fusion conformation (post-F). Compared with post-F, pre-F is more immunogenic. However, due to its instability, the pre-F protein expressed in vitro is easily converted to the post-fusion conformation, and the induced antibody neutralization activity is poor. The immune effect is not good in clinical trials. Therefore, solving the conformational stability of pre-F has been the focus and difficulty of RSV vaccine development for decades.

[0003] The first recombinant protein RSV vaccine to enter the clinical stage comes from Novavax. The immunogen is a modified F protein with a unique "pre-fusogenic" conformation, which is an intermediate state between pre-fusion and post-fusion. However, the Phase III clinical trials of this vaccine in the elderly and pregnant women did not reach the expected endpoints. Relevant analysis showed that the vaccine failed to stimulate sufficient neutralizing antibodies, which may be related to the fact that the conformation of its F protein is not completely pre-fusion.

[0004] Currently, there is no RSV vaccine on the market in China, but three vaccines have been launched overseas. Among them, GSK's AREXVY and Pfizer's ABRYSVO are recombinant protein vaccines, both of which are freeze-dried preparations. The results of relevant stability studies have shown that in liquid dosage forms, the conformation of pre-F will change significantly after being stored at 2-8°C for one month, affecting the immunogenicity of the protein.

[0005] However, the production process of freeze-dried preparations is complex, costly, and the medication steps are cumbersome. If the pre-fusion conformation of the pre-F protein can be kept stable in a liquid dosage form, it will not only simplify the vaccination steps, but also greatly reduce production costs and improve the accessibility of RSV vaccines.

[0006] Adjuvants have always been an important component of vaccines, among which aluminum adjuvants are the most widely used. However, studies have found that aluminum adjuvants cannot effectively activate the body's cellular immunity. The protective effect of cellular immunity has received increasing attention in recent years. Therefore, there is an urgent need to develop new adjuvants or adjuvant combinations that can induce better cellular immunity while activating humoral immunity.

[0007] Therefore, the development of a new adjuvant combination and a fully liquid RSV vaccine based on the adjuvant combination is of great significance for protecting the human body from RSV virus infection. Summary of the invention

[0008] In order to solve the above problems existing in the prior art, the first aspect of the present invention provides an adjuvant composition, which includes MF59 and CpG.

[0009] The second aspect of the present invention provides a fully liquid RSV vaccine, wherein the fully liquid RSV vaccine contains RSV pre-F protein and the adjuvant composition provided by the first aspect of the present invention.

[0010] The third aspect of the present invention provides a method for preparing a fully liquid RSV vaccine, the method comprising the operation of combining the RSV pre-F protein with the adjuvant composition provided by the first aspect of the present invention.

[0011] The adjuvant composition and the all-liquid RSV vaccine based on the adjuvant composition provided by the present invention can maintain the stability of the pre-fusion conformation of the pre-F protein in the liquid dosage form, while improving the cellular immune effect and humoral immune effect of the vaccine, which can greatly reduce the preparation cost of the vaccine and improve the accessibility of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The ELISA method in Example 3 was used to determine the titer of specific IgG antibodies in the serum of each group of mice after the second immunization.

[0013] Figure 2 The ELISA method in Example 3 was used to determine the titer of specific IgG1 antibodies in the serum of each group of mice after the second immunization.

[0014] Figure 3 The ELISA method in Example 3 was used to determine the titer of specific IgG2a antibodies in the serum of each group of mice after the second immunization.

[0015] Figure 4 This is the Elispot method used in Example 3 to specifically analyze the level of IFN-γ in the spleen cells of each group of mice after the second immunization under the stimulation of the RSV F protein peptide library.

[0016] Figure 5 The immunofluorescence inhibition method in Example 3 was used to detect the neutralizing antibody titer against RSV A2 virus in the serum of each group of mice after the second immunization. DETAILED DESCRIPTION

[0017] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0018] The first aspect of the present invention provides an adjuvant composition, which is particularly suitable for preparing a full liquid formulation RSV vaccine.

[0019] According to the first aspect of the present invention, the adjuvant composition comprises two adjuvants, MF59 and CpG.

[0020] The CpG (oligodeoxynucleotide) adjuvant may be type A, type B or type C, preferably type B, and more preferably CpG1018.

[0021] In the adjuvant composition provided in the first aspect of the present invention, the concentration of MF59 is 250 μL / mL-600 μL / mL, for example, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, The range of any one or two of 540, 550, 560, 570, 580, 590, 600 μL / mL, for example, 480-520 μL / mL, 460-540 μL / mL, 450-550 μL / mL, 420-530 μL / mL, 400-550 μL / mL, 300-540 μL / mL, 250-560 μL / mL, 280-520 μL / mL, preferably 500 μL / mL.

[0022] The concentration of CpG1018 is 50 μg / mL-500 μg / mL, for example, it can be any one of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 180, 200, 220, 250, 270, 300, 320, 350, 380, 400, 420, 450, 480, 500 μg / mL or a range between two thereof, for example, any one of 90-110 μg / mL, 80-120 μg / mL, 70-130 μg / mL, 60-140 μg / mL, 50-150 μg / mL, 90-200 μg / mL, 80-300 μg / mL, 90-400 μg / mL, 70-500 μg / mL, preferably 100 μg / mL.

[0023] The second aspect of the present invention provides a fully liquid RSV vaccine, wherein the fully liquid RSV vaccine contains RSV pre-F protein and the adjuvant composition provided by the first aspect of the present invention.

[0024] According to the second aspect of the invention, in the all-liquid RSV vaccine, the concentration of the RSV pre-F protein in the all-liquid RSV vaccine is 120-480 μg / mL, for example, a range between any one or two of 120, 150, 180, 200, 220, 240, 250, 280, 300, 320, 350, 380, 400, 420, 450, and 480 μg / mL, for example, 220-280 μg / mL, 200-300 μg / mL, 180-320 μg / mL, 150-350 μg / mL, 120-400 μg / mL, 200-400 μg / mL, 180-450 μg / mL, and 240-480 μg / mL, and preferably 240 μg / mL.

[0025] According to the second aspect of the present invention, the all-liquid RSV vaccine may further contain pharmaceutical excipients, which may include at least one of a buffer, sodium chloride, and a disaccharide.

[0026] Wherein, the buffer may be a citric acid / citrate buffer. The buffer is used to maintain the pH stability of the all-liquid RSV vaccine. The citrate may be sodium citrate and / or potassium citrate.

[0027] Among them, the concentration of citric acid / citrate in the full liquid formulation RSV vaccine is 5mM-20mM (calculated as citrate), for example, any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20mM or a range between two thereof, for example, 9-11mM, 8-12mM, 7-13mM, 6-14mM, 5-15mM, 11-29mM, 10-18mM, preferably 10mM.

[0028] Wherein, sodium chloride is used to maintain the physiological osmotic pressure suitable for the full liquid preparation RSV vaccine. The concentration of sodium chloride in the full liquid preparation RSV vaccine can be 0-200mM, such as 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200mM, any one or two of the range, such as 120-180mM, 120-200mM, 120-160mM, 100-140mM, 20-140mM, 40-160mM, preferably 140mM.

[0029] Wherein, disaccharide is used to maintain the physiological osmotic pressure suitable for the full liquid preparation RSV vaccine. Disaccharide can be at least one of sucrose, fucose, trehalose, etc. The concentration of disaccharide in the adjuvant composition is 0-300mM, such as 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300mM, any one or two of them, such as 200-240mM, 180-260mM, 160-280mM, 80-240mM, 40-200mM, 20-240mM, preferably 220mM.

[0030] According to the second aspect of the invention, the pH of the all-liquid RSV vaccine is 6.0-7.0, for example, in the range of any one or two of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, for example, 6.0-6.4, 6.1-6.3, 6.2-6.5, 6.2-6.8, 6.7-7.0, preferably 6.2.

[0031] According to the second aspect of the present invention, the unit dose of the all-liquid RSV vaccine may be 0.5 mL (500 μL), that is, the vaccine may be packaged at 0.5 mL per dose for ease of transportation and use.

[0032] The third aspect of the present invention provides a method for preparing a fully liquid RSV vaccine, the method comprising the operation of combining the RSV pre-F protein with the adjuvant composition provided by the first aspect of the present invention.

[0033] According to the third aspect of the invention, in the all-liquid RSV vaccine, the concentration of the RSV pre-F protein in the all-liquid RSV vaccine is 120-480 μg / mL, such as 120, 150, 180, 200, 220, 240, 250, 280, 300, 320, 350, 380, 400, 420, 450, 480 μg / mL, any one or a range between two of them, such as 220-280 μg / mL, 200-300 μg / mL, 180-320 μg / mL, 150-350 μg / mL, 120-400 μg / mL, 200-400 μg / mL, 180-450 μg / mL, 240-480 μg / mL, preferably 240 μg / mL.

[0034] According to the third aspect of the present invention, the method comprises diluting the RSV pre-F protein stock solution, adding the CpG1018 stock solution, and then adding MF59 after constant volume, and mixing.

[0035] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.

[0036] Example 1

[0037] This example is used to illustrate that the all-liquid preparation RSV vaccine provided by the present invention has high stability.

[0038] Under the premise of keeping the RSV pre-F protein content (240 μg / mL pre-F protein) and adjuvant content consistent (500 μL / mL MF59, 100 μg / mL CpG1018), the following five groups of prescriptions shown in Table 1 were designed:

[0039] Table 1 Five groups of all-liquid RSV vaccine prescriptions

[0040] Group pH Citric acid / sodium citrate concentration Sodium chloride concentration Sucrose concentration 1 6.0 10mM 140mM / 2 6.2 10mM 140mM / 3 6.5 20mM 125mM / 4 6.5 10mM / 220mM 5 7.0 10mM / 220mM

[0041] The above five groups of prescriptions were subjected to accelerated stability experiments at 40°C for 4 weeks to investigate the purity (purity refers to the proportion of the main peak of RSV pre-F protein trimer detected by SEC-HPLC) and activity of pre-F protein (AM14 antibody can bind to the trimer conformation of RSV pre-F protein, but not to post-F protein. The strength of the binding of AM14 antibody to pre-F protein can be measured by ELISA to characterize the pre-fusion conformation of pre-F protein. References: Gilman MSA, Moin SM, Mas V, Chen M, Patel NK, Kramer K, et al. (2015) Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PLoS Pathog 11(7):e1005035.doi:10.1371 / journal.ppat.1005035) changes, the specific data are shown in Table 2 below:

[0042] Table 2 Results of accelerated experiments for five groups of prescriptions

[0043]

[0044] From the results shown in Table 2, it can be seen that in the accelerated experiment at 40°C and 4 weeks, the purity and activity of the pre-F protein of the five groups of prescriptions did not change significantly, indicating that the pre-fusion conformation of the pre-F protein in the all-liquid RSV vaccine provided by the present invention has good stability and can be developed into a liquid preparation, especially the second group of prescriptions has the highest stability.

[0045] Example 2

[0046] This example is used to illustrate the preparation of a fully liquid formulation RSV vaccine.

[0047] Taking the preparation of 1000 mL of finished product liquid as an example, the thawed RSV pre-F protein stock solution was taken, mixed, the protein content was measured, and the protein stock solution was diluted to 1000 μg / mL with stock solution buffer (containing 0.13 g / L citric acid, 2.76 g / L sodium citrate) to obtain a diluted protein stock solution.

[0048] Use the preparation diluent (containing 2.941 g / L sodium citrate and 31.47 g / L sodium chloride) to dissolve the CpG1018 lyophilized powder. Use 800 mL of the preparation diluent to dissolve every 1 g of CpG1018 lyophilized powder. After filtration, obtain the CpG1018 mother liquor and detect the CpG1018 content in the CpG1018 mother liquor.

[0049] Take 240g of diluted protein stock solution and add CpG1018 mother solution to make the CpG1018 content in the final adjuvant-containing liquid be 100μg / mL. Then use the diluted preparation solution to make the volume to 500g, mix well, and then add 500g of MF59. After mixing, sterilizing and filtering, the filling machine is filled into penicillin bottles (0.5mL each), and the filled samples are labeled and outsourced to obtain the recombinant respiratory syncytial virus vaccine full liquid preparation, in which the RSV pre-F protein content is 240μg / mL, the concentration of MF59 is 500μL / mL, the concentration of CpG1018 is 100μg / mL, the concentration of citric acid / sodium citrate is 10mM, the pH is 6.2, and the concentration of sodium chloride is 140mM.

[0050] Example 3

[0051] This example is used to illustrate the mouse immunization experiment of recombinant respiratory syncytial virus vaccines prepared with different adjuvants, and the detection experiment of neutralizing antibodies against RSV virus in mouse serum.

[0052] Table 3 Composition of recombinant respiratory syncytial virus vaccines with different adjuvants

[0053]

[0054] Immunization method: After gently shaking each vaccine sample in Table 3 before use, use a disposable sterile syringe and needle to extract 50 μL of the vaccine sample solution and inject it into the outer thigh muscles of mice (5 mice were used for each vaccine group). Immunization time: Administer twice at an interval of 21 days, each time 50 μL.

[0055] Serum collection: Blood was collected from experimental animals in each test group on the 35th day after the first immunization, and serum was separated. The serum-specific IgG antibody and its antibody subtype titer were determined by enzyme-linked immunosorbent assay (ELISA). Mouse spleens were separated 49 days after the first immunization, and IFN-γ was detected by ELISPOT method. The neutralizing activity of animal serum against RSV virus 14 days after the second immunization was detected by immunofluorescence method.

[0056] 1. ELISA specific IgG antibody detection

[0057] The RSV Pre-F protein (antigen) (purchased from ACROBiosystems Group with the product number RSF-V52H7) was diluted to 4 μg / mL with coating solution (PBS buffer), and 100 μL / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution (PBS+0.05% Tween 20), 200 μL of blocking solution (PBS+1% BSA) was added to each well and blocked at 25°C for 1 hour. After blocking, the blocking solution was poured out, and the immunized mouse serum was diluted in multiples and added to the ELISA plate, 100 μL / well, and incubated at 25°C for 2 hours. After washing 3 times with washing solution, HRP-labeled goat anti-mouse IgG (1:2500) was added, 100 μL / well, and incubated at 25°C for 1 hour. After washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was reacted at 25°C in the dark for 15 minutes, and 100 μL of stop solution (2MH2SO4) was added to terminate the reaction. The OD450 value of each well was measured with an ELISA instrument. The dilution corresponding to the well whose OD450 value was 2.1 times greater than the OD450 value of the blank well (i.e., the control well incubated with PBS) was determined as the titer of the sample.

[0058] 2. Specific IgG antibody subtype titer detection

[0059] The RSV Pre-F protein was diluted to 4 μg / mL with coating solution (PBS buffer), and 100 μL / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution (PBS+0.05% Tween 20), 200 μL of blocking solution (PBS+1% BSA) was added to each well and blocked at 25°C for 1 hour. After blocking, the blocking solution was poured out, and the immunized mouse serum was diluted in multiples and added to the ELISA plate, 100 μL / well, and incubated at 25°C for 2 hours. After washing 3 times with washing solution, biotin-labeled sheep anti-mouse IgG1 (1:4000) and IgG2a (1:2500) were added, 100 μL / well, and incubated at 25°C for 1 hour. After washing 3 times with washing solution, add HRP-Labeled Streptavidin (1:10000), wash 5 times with washing solution, add TMB substrate colorimetric solution, react for 15 minutes at 25°C in the dark, and add 100 μL stop solution (2M H2SO4) to terminate the reaction. Measure the OD450 value of each well with an ELISA reader. The dilution corresponding to the well with an OD450 value greater than 2.1 times the OD450 value of the blank well (i.e., the control well incubated with PBS) is defined as the titer of the sample.

[0060] The results are as follows Figure 1-3 As shown by Figure 1 It can be seen that the ELISA method was used to measure the specific IgG antibody titers in the serum of each group of mice after the second immunization, which showed that the IgG antibody titers of all adjuvant-containing groups (Groups 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14) were significantly higher than those of the group without adjuvant (Group 1), among which the IgG antibody titers of the groups containing double adjuvant MF59+CpG1018 (Groups 5, 6, 7, 8, 10, 11, 12, 13, and 14) were higher than those of the aluminum hydroxide adjuvant group (Group 2), the CpG1018 single adjuvant group (Group 3), and the MF59+CpG1018 double adjuvant group (Group 4). adjuvant group (group 3), MF59 single adjuvant group (groups 4 and 9); when the CpG1018 content was the same, the IgG antibody titers of the groups with MF59 content of 500 μL / mL (groups 4, 5, 6, 7, and 8) were all higher than those of the groups with MF59 content of 250 μL / mL (groups 9, 10, 11, 12, and 13); and when the MF59 content continued to increase to 550 μL / mL (group 14), the IgG antibody titer was lower than that of the group with MF59 content of 500 μL / mL in the 6th group; Figure 2 It can be seen that the changing trend of IgG1 antibody titer in each group is basically consistent with that of IgG antibody titer; Figure 3It can be seen that the change trend of IgG2a antibody titer in each group is basically consistent with that of IgG and IgG1 antibody titers. The IgG2a antibody titer of each group containing dual adjuvant MF59+CpG1018 (Groups 5, 6, 7, 8, 10, 11, 12, 13, and 14) was significantly higher than that of the four single adjuvant groups (Groups 2, 3, 4, and 9), indicating that the immune response induced by the dual adjuvant group is Th1-biased. According to relevant literature reports, Th1-biased immune response is more conducive to preventing RSV virus infection and reducing the risk of "vaccine enhanced disease" (VED). Moreover, with the increase of CpG1018 and MF59 content, the IgG, IgG1 and IgG2a antibody titers did not increase linearly, but reached the highest when the CpG1018 content was 100μg / mL and the MF59 content was 500μL / mL, indicating that there is an optimal dose of MF59 and CpG1018, that is, the MF59 content is 500μL / mL and the CpG1018 content is 100μg / mL.

[0061] 3. ELISPOT method for detection of IFN-γ

[0062] After collecting the mouse spleen, store it in an EP tube containing 4mL RPMI 1640 culture medium (Gibico, 61870127) and grind it in a biosafety cabinet to release the cells in the spleen. Use a pipette to transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 4°C (1500rpm, 5min). Collect the cells and add preheated red blood cell lysis buffer. After mixing, place the cells in a 37°C incubator for 5min and centrifuge again at 4°C (1500rpm, 5min). Collect the cells and add serum-free culture medium containing 1% PS to mix, and centrifuge at 4°C (1500rpm, 5min). After collecting the cells, add serum-free culture medium containing 1% PS to mix, and centrifuge at 4°C (1500rpm, 5min). After collecting the cells, add RPMI 1640 culture medium (Gibico, 61870127) to mix, resuspend and count. After counting, adjust the cell density to 1×10 6 cells / mL. Add the stimulant to the ELISPOT plate coated with the capture antibody at 30 μL / well, with 2 replicates for each sample. 6After mixing cells / mL cell suspension, add 170μL / well to the well plate by vertical and slow dripping. Place the cell plate horizontally in the incubator and incubate for 38h before starting the test. Take out the ELISPOT plate from the incubator, discard the cells, and wash 5 times with PBS at 200μL / well. Add 100μL / well of detection antibody and incubate at room temperature for 2h. Discard the incubation solution, wash 5 times with PBS, add 100μL / well of Streptavidin-HRP working solution and incubate at room temperature for 1h. Discard the incubation solution, wash 5 times with PBS, add 100μL / well of TMB colorimetric solution until obvious spots appear, discard the colorimetric solution, and wash the well plate with distilled water. Remove the bottom cover plate, wait for the well plate to dry, and take pictures and read with a CTL plate reader.

[0063] The results are as follows Figure 4 As shown by Figure 4 It can be seen that the level of specific analysis of IFN-γ in the spleen cells of each group of mice after the second immunization under the stimulation of RSV F protein peptide library determined by the Elispot method showed that the single protein group (Group 1), aluminum hydroxide adjuvant group (Group 2) and CpG1018 single adjuvant group (Group 3) did not induce obvious specific cellular immunity, and the MF59 single adjuvant group (Groups 4 and 9) and MF59+CpG1018 double adjuvant group (Groups 5, 6, 7, 8, 10, 11, 12, 13, and 14) were able to induce obvious cellular immune response. However, with the increase of CpG1018 and MF59 content, the level of specific analysis of IFN-γ did not increase linearly, but when the CpG1018 content was 100 μg / mL and the MF59 content was 500 μL / mL, the cellular immune response was the strongest, which was consistent with the trend of the above-mentioned binding antibody titer.

[0064] 4. Detection of neutralizing antibodies against RSV A2 virus in mouse serum

[0065] The day before the experiment, cells were plated in a 96-well plate, with 3 × 10 cells per well. 4 cells / well. Culture for about 16 hours. Dilute the serum sample to be tested in multiples using serum-free medium and add 2×10 3After the virus amount of IFU / well is shaken and mixed, it is incubated in a 37℃ incubator for 1 hour. Discard the cell culture medium, add 100μL / well serum-virus mixture to the 96-well plate, and infect for 2 hours at 37℃. Discard the infection solution, add 100μL low serum medium (DMEM-F12 containing 2% FBS) to each well, and culture in a 37℃, 5% CO2 incubator for about 20h. Discard the culture medium in the plate in the P2 biosafety cabinet, add 200μL 4% PFA fixative to each well, open the lid and irradiate with ultraviolet light for 1h. Discard the fixative, add 100μL / well of membrane permeabilization solution to the cell plate and incubate at room temperature for 0.5h, discard the solution, wash twice with the cleaning solution, and pat dry gently. 10% FBS+PBS, 200μL / well, incubate at room temperature for 0.5h, discard the solution, wash twice with the cleaning solution, and pat dry gently. D25 antibody (Cambridge Biologics, LLC, 01-07-0120) was diluted with antibody diluent, added at 100 μL / well, incubated at room temperature for 1 hour, discarded, washed 5 times with cleaning solution, and patted dry. Diluted goat anti-human IgG-AF488 (Invitrogen, A-11013) was added, incubated at room temperature in the dark for 1 hour, discarded, washed 5 times with cleaning solution, and patted dry. The ELISA spot analyzer was used to take pictures and count, and the PRNT50 of the serum (serum dilution that reduces the fluorescent spots caused by the virus by 50%) was calculated.

[0066] The PRNT50 of the serum (serum dilution that reduces the fluorescent spots caused by the virus by 50%) was calculated.

[0067] The results are as follows Figure 5 As shown by Figure 5 It can be seen that the neutralizing antibody titer against RSV A2 virus in the serum of each group of mice after the second immunization was detected by immunofluorescence inhibition method, which showed that the neutralizing antibody levels of the single protein group (Group 1), aluminum hydroxide adjuvant group (Group 2) and CpG1018 single adjuvant group (Group 3) were all low, and the neutralizing antibody levels of the MF59 single adjuvant group (Groups 4 and 9) and MF59+CpG1018 double adjuvant group (Groups 5, 6, 7, 8, 10, 11, 12, 13, and 14) were all high. However, with the increase of CpG1018 and MF59 content, the neutralizing antibody level did not increase linearly, but was highest when the CpG1018 content was 100 μg / mL and the MF59 content was 500 μL / mL, which was consistent with the trend of the above-mentioned binding antibody titer and IFN-γ level.

[0068] Based on the above results, it can be seen that the vaccine containing dual adjuvants of MF59+CpG1018, especially when the CpG1018 content is 100μg / mL and the MF59 content is 500μL / mL, can produce a strong specific immune response in BALB / c mice.

[0069] In summary, the recombinant respiratory syncytial virus vaccine provided by the present invention is obtained by adding a specific amount of MF59+CpG1018 double adjuvants to the recombinant RSV pre-F protein stock solution to obtain a liquid dosage form of the recombinant respiratory syncytial virus vaccine. The immune experimental study of intramuscular injection of BALB / c mice proved that the recombinant respiratory syncytial virus vaccine has good immunogenicity.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are within the protection scope of the present invention.

Claims

1. An adjuvant composition, characterized in that The adjuvant composition includes MF59 and CpG.

2. The adjuvant composition according to claim 1, characterized in that: CpG is type A, type B or type C, preferably CpG1018; The concentration of MF59 is 250 μL / mL-600 μL / mL; The concentration of CpG1018 is 50 μg / mL-500 μg / mL.

3. The adjuvant composition according to claim 2, characterized in that: The concentration of MF59 is any one of 480-520 μL / mL, 460-540 μL / mL, 450-550 μL / mL, 420-530 μL / mL, 400-550 μL / mL, 300-540 μL / mL, 250-560 μL / mL, and 280-520 μL / mL; The concentration of CpG1018 is any one of 90-110 μg / mL, 80-120 μg / mL, 70-130 μg / mL, 60-140 μg / mL, 50-150 μg / mL, 90-200 μg / mL, 80-300 μg / mL, 90-400 μg / mL, and 70-500 μg / mL.

4. The adjuvant composition according to claim 1, characterized in that: The concentration of MF59 was 500 μL / mL; The concentration of CpG1018 was 100 μg / mL.

5. A fully liquid RSV vaccine, characterized in that: The full liquid preparation RSV vaccine contains RSV pre-F protein and the adjuvant composition according to any one of claims 1-4.

6. The all-liquid RSV vaccine according to claim 5, characterized in that: The concentration of the RSV pre-F protein in the all-liquid RSV vaccine is 120-480 μg / mL; Preferably, the concentration of the RSV pre-F protein in the all-liquid RSV vaccine is any one of 220-280 μg / mL, 200-300 μg / mL, 180-320 μg / mL, 150-350 μg / mL, 120-400 μg / mL, 200-400 μg / mL, 180-450 μg / mL, and 240-480 μg / mL; Preferably, the concentration of the RSV pre-F protein in the all-liquid RSV vaccine is 240 μg / mL.

7. The all-liquid RSV vaccine according to claim 5 or 6, characterized in that: The all-liquid RSV vaccine also contains pharmaceutical excipients; The pharmaceutical excipients include at least one of a buffer, sodium chloride and disaccharides.

8. The all-liquid RSV vaccine according to claim 7, characterized in that: The buffer is citric acid / citrate buffer; The disaccharide is at least one of sucrose, fucose and trehalose.

9. The all-liquid RSV vaccine according to claim 8, characterized in that: The pH of the all-liquid RSV vaccine is 6.0-7.0; Preferably, the pH of the all-liquid RSV vaccine is 6.

2.

10. A method for preparing a fully liquid RSV vaccine, the method comprising the step of mixing RSV pre-F protein with the adjuvant composition according to any one of claims 1 to 4.

Images