Stable recombinant respiratory syncytial virus pre-fusion F protein vaccine composition

Abstract

The invention discloses a stable F protein vaccine composition before fusion of a recombinant respiratory syncytial virus. According to the vaccine composition provided by the invention, a stable pre-fusion trimer structure can be maintained in the storage process of the recombinant pre-fusion F protein, and the combination with D25, AM14 and other antibodies with efficient RSV virus neutralizing activity can be maintained; besides, the vaccine composition can induce a high-titer binding antibody and a high-titer neutralizing antibody on a mouse model under the condition that a vaccine adjuvant is not added, and the lung tissue virus load can be remarkably reduced in a challenge experiment. Therefore, the vaccine composition has a good immune protection effect on the respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a stable recombinant respiratory syncytial virus pre-fusion F protein vaccine composition. Background Art

[0002] Respiratory syncytial virus (RSV) is a common respiratory viral infection that threatens health and life. RSV causes about 3.4 million hospitalizations and at least 66,000 deaths each year. Infants and preschoolers under 2 years old are the main populations infected by RSV. Almost all children have been infected with RSV before the age of 2 and will be repeatedly infected.

[0003] RSV is a negative-sense single-stranded RNA virus of the genus Pneumovirus in the family Paramyxoviridae. The glycoprotein (G protein) and fusion protein (F protein) encoded by its genes are the main antigens for vaccine design. The G protein is responsible for binding to the receptor, and the F protein is responsible for fusing the virus with the host cell membrane. The F protein irreversibly transforms from an unstable pre-fusion conformation to a stable post-fusion conformation. In studies of organisms naturally infected with RSV, it was found that antibodies with highly efficient neutralizing antibody activity mainly recognize the pre-fusion conformation, and some antibodies only recognize the pre-fusion protein structure, but not the post-fusion protein structure. Therefore, the pre-fusion conformation is the main direction of recombinant F protein vaccine design.

[0004] Because the structure of the F protein before fusion is unstable, its conformation has a tendency to refold into a post-fusion conformation in the solution, and it is necessary to design and develop an RSV F protein that can be stabilized in the pre-fusion conformation so that it can maintain a stable pre-fusion conformation. The applicant obtained a series of F proteins that can be stabilized in the pre-fusion conformation by designing F protein mutants in the early stage. However, in the long-term storage process, various physical and chemical degradations, such as aggregation and fracture, will still occur. These physical and chemical degradations will not only cause the antigen to be unable to maintain a stable conformation, but may even cause the protein to form a precipitation, resulting in a reduction in protein content. Therefore, it is necessary to develop a vaccine composition for the F protein that can stabilize the pre-fusion conformation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a stable recombinant respiratory syncytial virus pre-fusion F protein vaccine composition, which can reduce the physicochemical degradation rate of the protein, maintain a stable conformation and content during long-term storage, and stimulate the body to produce high-titer binding antibodies and neutralizing antibodies.

[0006] The first object of the present invention is to provide a stable recombinant respiratory syncytial virus pre-fusion F protein vaccine composition, wherein the pH of the vaccine composition is 5.9-8.3, and the vaccine composition comprises:

[0007] i) recombinant respiratory syncytial virus prefusion F protein;

[0008] ii) 5-30 mM buffer;

[0009] iii) 50-300 mM stabilizer;

[0010] iv)-0.1-1.0g / L surfactant

[0011] Wherein, the buffer is citric acid-sodium citrate buffer salt; the stabilizer is one or more of trehalose, sucrose or arginine hydrochloride.

[0012] Furthermore, in the vaccine composition, the content of the recombinant respiratory syncytial virus pre-fusion F protein is 20-1000 μg / mL, and the content of each vaccine dose is 10-500 μg / dose.

[0013] Furthermore, the stabilizer may also contain one or both of disodium edetate and aspartic acid.

[0014] Furthermore, the pH of the vaccine composition is 6.0-7.5.

[0015] Furthermore, the surfactant is one or more of polysorbate 20, polysorbate 80, and poloxamer 188.

[0016] Furthermore, the concentration of the surfactant is 0.1-0.5 g / L.

[0017] Furthermore, the vaccine composition may also contain a pharmaceutically acceptable carrier.

[0018] Furthermore, the carrier is an aluminum adjuvant, a nucleic acid adjuvant, a liposome adjuvant, an emulsion adjuvant or a composite adjuvant.

[0019] Furthermore, the vaccine composition is a liquid preparation or a lyophilized powder.

[0020] Furthermore, the vaccine composition is administered by intramuscular injection, subcutaneous injection, nasal spray, aerosol inhalation or oral administration.

[0021] The vaccine composition of the present invention has the following characteristics:

[0022] i) The vaccine composition can be stored stably in liquid state at 2-8 degrees Celsius for at least 4 weeks

[0023] ii) the vaccine composition can be stably stored in a liquid state at 25 degrees Celsius for at least 4 weeks;

[0024] iii) the vaccine composition can be stored stably at 40 degrees Celsius for at least 4 weeks in a freeze-dried state;

[0025] iv) the vaccine composition (without adjuvant) is capable of producing high titers of binding antibodies and neutralizing antibodies in BALB / c mice;

[0026] v) The vaccine composition (without adjuvant) significantly reduced the viral load inside lung tissue in BALB / c mice.

[0027] vi) The binding antibody and neutralizing antibody titers produced in BALB / c mice can be further increased when the vaccine composition contains an adjuvant.

[0028] By means of the above scheme, the present invention has at least the following advantages:

[0029] The recombinant respiratory syncytial virus vaccine composition provided by the present invention can maintain a stable pre-fusion trimer structure of the recombinant pre-fusion F protein during storage, and can maintain binding with antibodies such as D25 and AM14 that have high RSV virus neutralizing activity; in addition, the vaccine composition of the present invention can induce high-titer binding antibodies and neutralizing antibodies in a mouse model without adding a vaccine adjuvant, and can significantly reduce the viral load in lung tissue. This shows that the vaccine has a good immune protection effect against respiratory syncytial virus.

[0030] The above description is only an overview of the technical solution and some results of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described as follows with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SEC profiles of freeze-dried samples after 2 and 4 weeks at 40°C / 75% RH;

[0032] Figure 2 Serum IgG binding antibody levels induced by different vaccine compositions;

[0033] Figure 3 Levels of neutralizing antibodies induced by different vaccine compositions against live wild-type A2 and B viruses;

[0034] Figure 4 Virus load in lung tissue of BALB / c mice 5 days after infection. DETAILED DESCRIPTION

[0035] The present invention discloses a stable recombinant respiratory syncytial virus prefusion F protein vaccine composition. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] Detection Methods

[0037] Size Exclusion Chromatography (SEC):

[0038] Using NanoChrom BioCore SEC-300 (5μm, 7.8*300mm) column, on Shimadzu LC-20A system, the mobile phase was 20mM Pb + 300mM NaCl, the flow rate was 0.5ml / min, isocratic elution was performed, and detection was performed by UV wavelength 280nm, and the percentage of peak area was used for quantitative purity.

[0039] Quantification of D25 and AM14 binding titers by thin film interferometry:

[0040] The binding titer of D25 and AM14 was quantified using the Gator label-free molecular interaction detector. The detection optical fiber pre-coated with protein A protein by the manufacturer was used to first bind to the D25 or AM14 antibody and then to the antigen. The binding rate between the antigen molecule and the antibody was positively correlated with the concentration. The antigen bound by the D25 or AM14 antibody was quantified using the standard curve.

[0041] Terms and Definitions

[0042] As used herein and in the appended claims, reference is made to the following definitions, unless the context clearly dictates otherwise.

[0043] Respiratory Syncytial Virus (RSV) is a common respiratory virus, especially in infants and young children. RSV infection usually presents as mild cold symptoms, such as runny nose, cough and fever, but for people with weak immunity, such as premature infants, the elderly and those with impaired immune systems, it may cause serious respiratory diseases such as pneumonia and bronchitis.

[0044] "Trimer" refers to the naturally occurring form of the F protein on the surface of RSV virus, which is composed of three protomers.

[0045] "Pre-fusion protein (preF protein)" refers to the form of F protein on the surface of the RSV virus before it fuses with the cell membrane. When it fuses with the cell membrane, the pre-fusion protein will fold into an energetically favorable post-fusion state.

[0046] "Pharmaceutically acceptable" means that the indicator is suitable for administration to a subject (e.g., a human or animal subject). Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 15th edition (1975) describes compositions and formulations (including diluents) suitable for pharmaceutical delivery of therapeutic and / or prophylactic compositions (including immunogenic compositions).

[0047] The term "composition" as used herein refers to a combination of a recombinant antigen, an immunostimulant or adjuvant, a buffer, a stabilizer or osmotic pressure regulator, and a surfactant.

[0048] "Adjuvant" means an agent that enhances the production of an immune response in a non-antigen-specific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) to which antigens are adsorbed; emulsions, including water-in-oil, oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like receptor agonists (particularly TLR2, TLR4, TLR7 / 8 and TLR9 agonists), and various combinations of such components.

[0049] "D25 and AM14 antibodies" refer to antibodies that can specifically bind to RSV preF protein. D25 can specifically bind to preF protein protomers and trimers; AM14 can specifically bind to preF protein trimers, but cannot bind to protomers. Therefore, the stability of the preF protein trimer conformation can be judged by the binding ability of D25 and AM14. These two antibodies can be obtained by the technical means in the public literature: Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein, 2015 by Gilman MS et al.

[0050] "Binding antibodies" herein refer to antibodies that can bind to preF protein and are produced by the body after the body is immunized with preF protein, and are usually measured using enzyme-linked immunosorbent assay (ELISA).

[0051] "Neutralizing antibodies" in this article refer to antibodies produced by the body after immunization with preF protein that can bind to RSV virus and block virus-infected cells.

[0052] The recombinant respiratory syncytial virus pre-fusion F protein used in the present application is obtained by replacing, deleting and / or inserting mutations in the amino acid sequence of the respiratory syncytial virus fusion F protein. Compared with the wild-type F protein, it can form a trimer structure and maintain a relatively stable fusion precursor state, and the yield is greatly improved when the target protein is expressed in mammalian cells, and the protein stability is excellent.

[0053] Specifically include deleting the fragment containing the feline protease cleavage site in the amino acid sequence of respiratory syncytial virus fusion F protein. In the present application, deleting the fragment containing the feline protease cleavage site can avoid the wild-type RSV F protein from being cleaved by feline protease and causing breakage, thereby maintaining the pre-fusion conformation of the F protein. The present invention does not introduce any site-directed mutagenesis of the feline protease cleavage site sequence, and does not need to introduce a heterologous connection sequence, and can maintain a higher sequence similarity compared to the wild-type sequence.

[0054] In the present application, the amino acid sequence of the respiratory syncytial virus fusion F protein is shown in SEQ ID NO. 1, and the mutant is a mutant in which 28 to 46 amino acids between positions 100 and 147 in the amino acid sequence are deleted. For example, 37 amino acids between positions 105 and 143 in the amino acid sequence are deleted (a deletion of 37 amino acids between positions 105 and 143).

[0055] In the present application, the mutant also includes mutating the amino acid that undergoes alpha helix penetration during the conformational transformation of the respiratory syncytial virus fusion F protein from the fusion precursor to the post-fusion body into proline, or arbitrarily inserting one or more prolines to block the conformational change of the respiratory syncytial virus fusion F protein from the fusion precursor to the fusion post-fusion body, so as to stabilize the respiratory syncytial virus fusion F protein in the fusion precursor state.

[0056] For example, the respiratory syncytial virus fusion F protein with an amino acid sequence as shown in SEQ ID NO.1 is subjected to at least one of the following mutations:

[0057] Mutate glutamine at position 210 to proline (Q210P);

[0058] Mutate serine 211 to proline (S211P);

[0059] mutating serine at position 213 to proline (S213P);

[0060] Mutate isoleucine at position 214 to proline (I214P);

[0061] Mutation of isoleucine at position 217 to proline (I217P);

[0062] One or more proline residues are inserted between amino acids 210 and 217, for example, one proline residue is inserted between amino acids 215 and 216 (S215insertP).

[0063] In the present application, it also includes mutating one or more charged amino acids in the amino acid sequence of the respiratory syncytial virus fusion F protein into polar amino acids, hydrophobic amino acids or aromatic amino acids to release the electrostatic repulsion in the respiratory syncytial virus fusion F protein and enhance the stability of the respiratory syncytial virus fusion F protein fusion precursor.

[0064] For example, the respiratory syncytial virus fusion F protein with an amino acid sequence as shown in SEQ ID NO.1 is subjected to at least one of the following mutations:

[0065] Mutation of glutamic acid at position 60 to alanine (E60A), glycine (E60G), serine (E60S), threonine (E60T), leucine (E60L), methionine (E60M), or phenylalanine (E60F);

[0066] Mutating the aspartic acid at position 194 to alanine (D194A), glycine (D194G), serine (D194S), threonine (D194T), leucine (D194L), methionine (D194M), or phenylalanine (D194F);

[0067] Mutation of aspartic acid at position 486 to glycine (D486G), phenylalanine (D486F), or cysteine ​​(D486C);

[0068] The aspartic acid at position 489 was mutated to glycine (D489G), phenylalanine (D489F), or cysteine ​​(D489C).

[0069] In the present application, when aspartic acid at position 486 and / or position 489 is mutated to cysteine, interchain disulfide bonds can be formed while releasing repulsion to further enhance the stability of the F protein trimer.

[0070] In the present application, it also includes mutating one or more small-volume amino acids in the amino acid sequence of the respiratory syncytial virus fusion F protein into larger-volume hydrophobic amino acids to fill the surface cavities of the respiratory syncytial virus fusion F protein in the fusion precursor conformation, thereby enhancing the stability of the respiratory syncytial virus fusion F protein fusion precursor.

[0071] For example, the asparagine at position 67 of the respiratory syncytial virus fusion F protein with an amino acid sequence as shown in SEQ ID NO. 1 is mutated to phenylalanine (D67F).

[0072] In the present application, it also includes mutating the 509th serine of the respiratory syncytial virus fusion F protein as shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid (S509D).

[0073] In the present application, it also includes mutating the leucine at position 512 of the respiratory syncytial virus fusion F protein with an amino acid sequence such as SEQ ID NO.1 to glutamine (S512Q), asparagine (S512N) or phenylalanine (S512F).

[0074] In the present invention, the mutation of amino acids at positions S509 and L512 can increase the interaction between trimers and thus maintain the stability of the trimers.

[0075] In the present application, it also includes mutating the 74th alanine and the 218th glutamic acid of the respiratory syncytial virus fusion F protein as shown in the amino acid sequence of SEQ ID NO.1 to cysteine ​​(A74C, E218C).

[0076] In the present application, the alanine at position 74 and the glutamic acid at position 218 were mutated to cysteine ​​to form a disulfide bond, which can increase the trimer stability.

[0077] In the following examples, YK001Ag226 (E60L, S213P, S509D, L512Q, and 37 amino acids missing between positions 105 and 143) is taken as an example to further illustrate the present invention:

[0078] Example 1: Investigating the effect of pH on the stability of preF protein

[0079] Different formulation samples (9 in total) with a pH range of 4.2-8.3 were prepared, and their compositions included 1 mg / mL RSV pre F protein, 10 mM sodium citrate-phosphate, 240 mM trehalose, and 0.2 g / L polysorbate 80. The 9 formulation samples were placed at 37° C. for 0 and 3 days, respectively.

[0080] The Gator molecular interaction instrument was used to quantify the RSV pre F protein that can bind to the D25 antibody and the AM14 antibody. As can be seen from Table 1, after being placed at 37°C for 3 days, when the pH was 4.2, 4.7, and 5.4, the binding of the RSV pre F protein to the D25 and AM14 antibodies dropped to less than 80% of that on day 0, and when the pH was 5.9-8.3, the binding to the D25 antibody was above 80%, and the binding to the AM14 antibody was basically above 80% (except for 79% at pH 8.3).

[0081] Table 1 Changes in the binding capacity of samples with different pH values ​​at 37°C with antibodies

[0082]

[0083]

[0084] After the samples of the 9 prescriptions were placed at 37°C for 3 days, the changes in protein purity were detected using high performance liquid chromatography and size exclusion chromatography. As shown in Table 2, the samples at pH 4.2, 4.7, and 5.4, after being placed at 37°C for 3 days, the decrease in main peak purity was significantly higher than that of the samples at pH 5.9-8.3.

[0085] Table 2 Changes in SEC main peak purity of samples with different pH values ​​at 37°C

[0086] Sample No. pH SEC main peak purity reduction value at 37℃ for 3 days 1 4.2 80.2％ 2 4.7 84.5％ 3 5.4 40.4％ 4 5.9 25.6％ 5 6.4 20.6％ 6 6.8 19.6％ 7 7.3 20.5％ 8 7.8 22.1％ 9 8.3 23.6％

[0087] The above data show that when placed at 37°C for 3 days, between pH 5.9-8.3, the preF protein can maintain good binding ability with D25 antibody and AM14 antibody and SEC purity.

[0088] Example 2: Effect of buffer type on RSV pre F protein stability

[0089] Seven formulation samples containing different buffers as shown in Table 3 were prepared, and the seven formulation samples were placed at 37° C. for 0 and 3 days, respectively.

[0090] Table 3 Prescription composition of different buffers

[0091]

[0092]

[0093] The Gator molecular interaction instrument was used to quantify the RSV pre F protein that can bind to the D25 antibody and the AM14 antibody. As shown in Table 4, after being placed at 37°C for 3 days, in the prescription samples containing histidine-histidine hydrochloride and trishydroxyaminomethane hydrochloride, the binding of RSV pre F protein to the AM14 antibody dropped to less than 80% of that on day 0, and in the prescription samples containing citric acid-sodium citrate and sodium phosphate, the binding of RSV pre F protein to the AM14 antibody was maintained above 80%.

[0094] Table 4 Changes in binding capacity of samples containing different buffers with antibodies at 37°C

[0095]

[0096] After the samples of the seven prescriptions were placed at 37°C for 3 days, the samples containing histidine-histidine hydrochloride and trishydroxyaminomethane hydrochloride produced obvious visible particles, while the samples containing citric acid-sodium citrate and sodium phosphate were clear in appearance. For the prescription samples containing citric acid-sodium citrate and sodium phosphate, the protein purity changes were detected by high performance liquid chromatography and size exclusion chromatography. As shown in Table 5, after the prescription samples containing sodium phosphate were placed at 37°C for 3 days, the decrease in the main peak purity was more than twice that of the prescription samples containing citric acid-sodium citrate.

[0097] Table 5 Changes in SEC main peak purity of samples containing different buffers at 37°C

[0098] Sample No. SEC main peak purity reduction value at 37℃ for 3 days 12 7.8％ 13 4.9％ 14 20.2％ 15 15.6％

[0099] The above data show that only when placed in the citric acid-citric acid system at 37°C for 3 days can the RSV pre F protein maintain a relatively stable binding ability with antibodies and maintain a high purity.

[0100] Example 3: Effect of stabilizer type on the stability of RSV pre F protein

[0101] Seven prescriptions containing different stabilizers as shown in Table 6 were prepared, and samples of the seven prescriptions were placed at 37° C. for 0 and 3 hours respectively, and then taken out for testing.

[0102] Table 6 Prescription composition of different stabilizers

[0103]

[0104] The Gator Molecular Interaction Analyzer was used to quantify the RSV pre F protein that could bind to the D25 antibody and the AM14 antibody. As shown in Table 7, after being placed at 37°C for 3 days, the RSV pre F protein could maintain good binding ability with the D25 and AM14 antibodies in the formulations containing trehalose, or trehalose and disodium edetate, or glycine, or arginine hydrochloride, or proline, or trehalose and aspartic acid, or sodium chloride.

[0105] Table 7 Changes in binding capacity of samples containing different stabilizers with antibodies at 37°C

[0106]

[0107] After the samples of the seven prescriptions were placed at 37°C for 3 days, the protein purity changes were detected by high performance liquid chromatography and size exclusion chromatography. As shown in Table 8, after being placed at 37°C for 3 days, the main peak purity of the prescription samples containing glycine and proline decreased more than that of other samples, and could not maintain a stable SEC purity. The main peak purity decrease value of the prescription containing sodium chloride was slightly higher than that of the prescription containing trehalose. In the prescription samples containing trehalose and arginine hydrochloride, preF protein can maintain a relatively stable purity. The prescriptions of trehalose and disodium edetate, or trehalose and aspartic acid can also maintain a relatively stable purity.

[0108] Table 8 Changes in SEC main peak purity of samples containing different stabilizers at 37°C

[0109]

[0110]

[0111] The above data show that when placed at 37°C for 3 days, RSV pre F protein can maintain good binding ability with D25 and AM14 antibodies and higher SEC purity in the formulations containing trehalose, or trehalose and disodium edetate, or arginine hydrochloride, or trehalose and aspartic acid.

[0112] Example 4: Trehalose or sucrose can maintain good stability of preF protein

[0113] The formulation samples containing 20 mM citric acid-sodium citrate, 240 mM trehalose or sucrose, 0.2 g / L polysorbate 80, and pH 6.5 as shown in Table 9 were prepared, and the samples of the two formulations were placed at 37° C. for 0 and 3 hours respectively and then taken out for detection.

[0114] Table 9 Prescription composition of different stabilizers

[0115]

[0116] The Gator Molecular Interaction Analyzer was used to quantify the RSV pre F protein that could bind to the D25 antibody and the AM14 antibody. As shown in Table 10, after being placed at 37°C for 3 days, the RSV pre F protein in the formulation samples containing sucrose and trehalose could maintain good binding ability with the D25 and AM14 antibodies.

[0117] Table 10 Changes in binding capacity of samples containing trehalose or sucrose with antibodies at 37°C

[0118]

[0119] After the formulation samples containing sucrose and trehalose were placed at 37°C for 3 days, 25°C for 4 weeks, and 5°C for 4 weeks, the changes in protein purity were detected using high performance liquid chromatography and size exclusion chromatography. As shown in Table 11, the formulation samples containing sucrose or trehalose could maintain a purity of more than 92%.

[0120] Table 11 Changes in the purity of the main peak of SEC at 5°C, 25°C, and 37°C for samples containing trehalose or sucrose

[0121]

[0122]

[0123] Example 5: Freeze-dried stability

[0124] The sample was prepared with a formula of 20 mM citric acid-sodium citrate, 240 mM sucrose, 0.2 g / L polysorbate 80, pH 6.5, and freeze-dried using a freeze-drying method known in the art. The freeze-dried sample was placed in a 40°C / 75% RH constant temperature and humidity chamber, and samples were taken for testing at 2 weeks and 4 weeks. The protein purity change was tested by high performance liquid chromatography and size exclusion chromatography. The results are as follows: Figure 1 After being placed at 40°C for 2 weeks and 4 weeks, the chromatographic peak shape did not change significantly, indicating that the preF protein can be stable at 40°C for at least 4 weeks in a freeze-dried state.

[0125] Example 6: Immunological evaluation of vaccines containing dual adjuvants and those without adjuvants

[0126] 1. Preparation of vaccine composition

[0127] The specific preparation method of CpG7909 and aluminum hydroxide adjuvant is: first adsorb CpG7909 onto aluminum hydroxide adjuvant (the content of aluminum hydroxide adjuvant here is essentially the content of aluminum element), then add recombinant pre-fusion F protein to the CpG7909 / aluminum hydroxide sample, and use it after sufficient adsorption. If not used immediately, store at 2-8°C.

[0128] 2. Mouse grouping and animal immunization strategy

[0129] 24 SPF female BALB / c mice (6 weeks old) were grouped. Each group had 6 or 9 mice, for a total of 3 groups. Group information is shown in Table 12. According to the grouping in Table 12, the mice were immunized by intramuscular injection into the inner thigh of the mice, and the injection dose was 1 / 10 of the intended human dose. Each mouse was immunized twice, with an immunization interval of 3 weeks.

[0130] Table 12 Mouse grouping and immunization strategy

[0131] Group Dosage / unit Number of animals 1 Negative control (buffer) 6 2 6 μg preF protein 9 3 6μg preF protein + 25μg CpG7909 + 50μg aluminum hydroxide 9

[0132] 3. Vaccine-induced antibody response detection

[0133] Two weeks after the second immunization, blood was collected from each group of mice and the supernatant was separated. The IgG antibody titer against preF protein in the serum was detected by ELISA ( Figure 2 ); serum neutralizing antibody titers were detected by neutralization experiments based on wild-type live viruses RSV A2 and RSV B ( Figure 3 ). Where LOD stands for lower limit of detection.

[0134] Compared with the negative control group (buffer group), the antigen group and the antigen and adjuvant combination group can induce a higher level of binding antibody titer. The antigen group alone can induce a higher level of binding antibody titer, and the addition of adjuvant can further increase the binding antibody titer. From the results of the neutralizing antibody titer of mouse serum in each group detected by the neutralization experiment based on the wild-type live virus RSV A2 and RSV B, the neutralizing antibodies induced by the antigen group and the antigen and adjuvant combination have a good neutralizing effect on the wild-type live virus RSV A2 and RSV B.

[0135] The above results indicate that the RSV recombinant preF protein vaccine and the vaccine and adjuvant combination have good immunogenicity and can produce high titer binding and neutralizing antibodies.

[0136] 4. Lung tissue viral load

[0137] Three weeks after the second immunization, mice were anesthetized and challenged intranasally with 50 μl of 2.66 × 10 10 5 days after the challenge, all mice were killed, lung tissues were dissected and homogenized for viral load determination (qRT-PCR) ( Figure 4 ). Where LOD stands for lower limit of detection.

[0138] Seven days after the virus attack, the viral load in the lung tissue of mice in the buffer group was 2.01×10 9 copies / mL, and the viral load in the lung tissue of the antigen group was 2.54×10 6 The viral load in the lung tissue of the antigen and adjuvant combination group was 2.72×10 6 The viral load decreased by 2.57 lg compared with the buffer group (p<0.01). The above results show that the RSV recombinant preF protein vaccine and the vaccine and adjuvant combination have a good immune protection effect against respiratory syncytial virus.

[0139] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A stable recombinant respiratory syncytial virus pre-fusion F protein vaccine composition, characterized in that: The pH of the vaccine composition is 5.9-8.3, and the vaccine composition comprises: i) recombinant respiratory syncytial virus prefusion F protein; ii) 5-30 mM buffer; iii) 50-300 mM stabilizer; iv) 0.1-1.0 g / L surfactant Wherein, the buffer is citric acid-sodium citrate buffer salt; the stabilizer is one or more of trehalose, sucrose or arginine hydrochloride.

2. The vaccine composition according to claim 1, characterized in that In the vaccine composition, the content of the recombinant respiratory syncytial virus pre-fusion F protein is 20-1000 μg / mL.

3. The vaccine composition according to claim 1, characterized in that The stabilizer may also contain one or both of disodium edetate and aspartic acid.

4. The vaccine composition according to claim 1, characterized in that The pH of the vaccine composition is 6.0-7.

5.

5. The vaccine composition according to claim 1, characterized in that The surfactant is one or more of polysorbate 20, polysorbate 80, and poloxamer 188.

6. The vaccine composition according to claim 5, characterized in that The concentration of the surfactant is 0.1-0.5 g / L.

7. The vaccine composition according to claim 1, characterized in that The vaccine composition may further comprise a pharmaceutically acceptable carrier.

8. The vaccine composition according to claim 7, characterized in that The carrier is an aluminum adjuvant, a nucleic acid adjuvant, a liposome adjuvant, an emulsion adjuvant or a composite adjuvant.

9. The vaccine composition according to claim 1, characterized in that The vaccine composition is a liquid preparation or a lyophilized powder.

10. The vaccine composition according to claim 1, characterized in that: The vaccine composition is administered by intramuscular injection, subcutaneous injection, nasal spray, aerosol inhalation or oral administration.

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