A combined vaccine containing pneumococcus and influenza virus, preparation method and application thereof

Abstract

The present invention relates to a combined vaccine comprising pneumococcus and influenza virus, a preparation method and application thereof. The pneumococcus comprises a multivalent pneumococcal capsular polysaccharide, and the polysaccharide comprises at least polysaccharides from serotypes 6A, 17A and 22F. The multivalent pneumococcus and influenza vaccine stock solution of hemagglutinin from influenza virus are combined to form a combined vaccine. Multiple pathogen antigens are used in combination without inhibitory effects on each other and can synergistically improve the immunogenicity of the vaccine. The broad-spectrum antigens can simultaneously stimulate the body to produce humoral or cellular immune protection efficacy against multiple subtypes of influenza virus and multiple pneumococcal serotypes.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and specifically relates to a combined vaccine comprising pneumococcus and influenza virus, a preparation method and application thereof. Background Art

[0002] Streptococcus pneumoniae (also known as pneumococcus) was first isolated from the sputum of patients by Louis Pasteur and GMSternberg in France and the United States in 1881. It is Gram-positive, with spear-shaped bacteria, arranged in pairs or short chains. Toxic strains have a capsule with a chemical composition of polysaccharides outside the bacteria. The main pathogenic substances of pneumococcus are pneumolysin and capsule. The capsule is antigenic and is the basis for the typing of Streptococcus pneumoniae. Pneumococcus is divided into 91 serotypes according to the different antigenicity of the capsule polysaccharide. This bacterium can cause lobar pneumonia, meningitis, bronchitis and other diseases. There are many multivalent pneumonia vaccines on the market, but they still cannot include all common serotypes, and the immune protection against some serotypes is not very good.

[0003] Influenza virus is the abbreviation of influenza virus. It belongs to the family of Orthomyxoviridae and is a single-stranded RNA membrane virus. Its RNA contains eight segments. The genetic structure of influenza virus allows it to continuously exchange gene segments and thus form advantageous mutations. According to the nucleoprotein and membrane surface protein antigens, it is divided into types A (A), B (B), C (C) and D (D). The outer layer of influenza virus has two different glycoproteins that form radial protrusions, namely hemagglutinin (H) and neuraminidase (N). Antigenic variation of influenza virus refers to the change in the structure of H and N antigens. Among them, humans are mainly infected with type A and type B. Type A influenza virus is most likely to mutate. The influenza pandemic is caused by the emergence of new subtypes of type A influenza virus or the reappearance of old subtypes. Type A influenza virus is divided into many subtypes according to the differences in H and N antigens. H can be divided into 18 subtypes (H1~H18) and N has 11 subtypes (N1~N11). Common type B influenza viruses include BY and BV. Influenza virus infection will cause host cell degeneration, necrosis and even shedding, resulting in mucosal congestion, edema and increased secretions, thereby causing nasal congestion, runny nose, sore throat, dry cough and other upper respiratory tract infection symptoms. When the virus spreads to the lower respiratory tract, it may cause bronchiolitis and interstitial pneumonia.

[0004] When influenza is prevalent in winter, pneumococcal infectious diseases can be seen almost at the same time. Bronchitis and pneumonia often occur at the same time or after influenza virus infection. At this time, the pathogen is mostly pneumococcus. Especially for the elderly, severe pneumonia caused by pneumococcus may occur after influenza, and even death. In such cases, the number of deaths due to the combination of the two has increased, so vaccine prevention of pneumococcal infectious diseases combined with influenza is very important. Summary of the invention

[0005] To solve the above problems, the present invention provides a multivalent pneumococcal vaccine, a preparation method and an application thereof in preparing a combined vaccine. Specifically,

[0006] In one aspect, the present invention provides a combination vaccine comprising a multivalent pneumococcus, wherein the combination vaccine comprises (1) a multivalent pneumococcal capsular polysaccharide, wherein the polysaccharide comprises at least polysaccharides from serotypes 6A, 17A and 22F, and (2) an influenza vaccine stock solution comprising hemagglutinin from an influenza virus.

[0007] Preferably, the polysaccharide further comprises one or more of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F.

[0008] Preferably, the multivalent pneumococcal vaccine comprises a 25-valent pneumococcal vaccine and the polysaccharides comprise polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17A, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0009] More preferably, the ratio of polysaccharides of each serotype is 1:1.

[0010] Further preferably, each type is 10-50 μg / ml, which can be any value or range within the above range, for example 12, 15, 20, 25, 30, 35, 40, 45 or 50 μg / ml.

[0011] Preferably, the polyvalent pneumococcus is derived from a polyvalent capsular polysaccharide stock solution.

[0012] In the influenza vaccine stock solution,

[0013] Preferably, the influenza virus includes at least one or more influenza virus subtypes, and more preferably, the influenza virus is from influenza A, B, C and / or D virus.

[0014] More preferably, the influenza virus includes at least influenza A virus, such as H1N1, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8, etc.

[0015] In a specific embodiment, the influenza virus includes at least H1N1 and H3N2 subtypes;

[0016] More preferably, the influenza virus includes influenza A and B viruses.

[0017] In a specific embodiment, the influenza virus includes at least H1N1, H3N2 subtypes, B BY and BV subtypes.

[0018] In the combined vaccine, the hemagglutinin content of each influenza virus subtype is 15-50 μg / ml, which can be any range or value within the above range, for example, it can be 15, 18, 20, 22, 25, 18, 30, 35, 40, 45, 48, or 50 μg / ml.

[0019] Preferably, any of the above-mentioned combination vaccines further comprises an adjuvant.

[0020] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant. In one or more embodiments of the present invention, the adjuvant is at least any one of aluminum hydroxide adjuvant (Al(OH)3 adjuvant), CpG1018 adjuvant (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., batch number 0210426), CpG-cjx1 adjuvant (our company), and CpG7909 adjuvant (our company).

[0021] Preferably, the mass ratio of the vaccine stock solution: adjuvant is 1:(40-60), which can be any range or any value within the above range, such as 1:40, 1:42, 1:45, 1:48, 1:50, 1:52, 1:55, 1:58, 1:60 and the like.

[0022] The adjuvant includes aluminum adjuvant and / or CpG adjuvant, and more preferably, the CpG adjuvant includes CpG1018 adjuvant, CpG-cjx1 and / or CpG7909 adjuvant.

[0023] Furthermore, the adjuvant may be Al(OH)3 (aluminum hydroxide) adjuvant.

[0024] Further, the adjuvant includes an aluminum adjuvant and a CpG double adjuvant. More preferably, in the double adjuvant, the mass ratio of the aluminum adjuvant to the CpG adjuvant may be (22-28): 1, specifically 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1 or 28: 1, preferably 25: 1. The aluminum adjuvant is AL(OH) 3 (aluminum hydroxide), and the CpG adjuvant is CpG-cjx1 adjuvant.

[0025] In the combination vaccine, the content of aluminum adjuvant can be 400-600 μg / ml, which can be any range or value within the above range, for example, it can be 400, 420, 450, 480, 500, 520, 550, 580, 600 μg / ml.

[0026] In the vaccine, the content of CpG adjuvant can be 10-30 μg / ml, which can be any range or value within the above range, for example, it can be 10, 12, 15, 18, 20, 23, 26, 28, 29, 30 μg / ml and the like.

[0027] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0028] Preferably, the vaccine also includes a vaccine delivery system, which can be a type of substance that can carry antigenic substances to the body's immune system and store and exert its antigenic effects for a long time. The vaccine delivery system described herein can be a saline gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano adjuvant vaccine delivery system.

[0029] Furthermore, the combination vaccine also includes one or more pharmaceutically acceptable carriers.

[0030] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0031] The vaccine for preventing infection of the present invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.

[0032] Another aspect of the present invention provides a method for preparing the above-mentioned combination vaccine, which comprises (I) a step of preparing a multivalent pneumococcal capsular polysaccharide, and (II) a step of preparing an influenza vaccine stock solution.

[0033] Preferably, the steps of preparing the multivalent pneumococcal capsular polysaccharide include 1) culturing pneumococci; and 2) purifying the pneumococcal capsular polysaccharide.

[0034] Preferably, the step 2) comprises: 2-1) inactivating the pneumococcal fermentation broth, collecting the supernatant by centrifugation, and concentrating it by ultrafiltration; 2-2) concentrating the concentrate by alcohol precipitation, acid precipitation and calcium precipitation, and collecting the supernatant after concentration; 2-3) filtering and sterilizing to obtain a polyvalent capsular polysaccharide stock solution.

[0035] More preferably, in step 2-2), ethanol is used for alcohol precipitation, acetic acid is used for acid precipitation, and / or calcium chloride solution is used for calcium precipitation.

[0036] More preferably, the multivalent capsular polysaccharide stock solution is stored at -20°C.

[0037] More preferably, the multivalent capsular polysaccharide stock solution includes 25 polysaccharide types which are mixed in a solid-to-total ratio of 1:1 and diluted to a certain concentration with phosphate buffer to prepare a 25-valent pneumococcal vaccine, wherein each type of 25-valent pneumococcal polysaccharide is 10-50 μg / ml, which can be any value or range within the above range, for example 12, 15, 20, 25, 30, 35, 40, 45 or 50 μg / ml.

[0038] Preferably, the steps of preparing the influenza vaccine stock solution include influenza virus amplification, harvesting, lysis and inactivation.

[0039] More preferably, after the harvest, the harvest liquid is clarified and filtered to obtain a clarified liquid, and the filtration includes primary filtration and fine filtration. Further preferably, the pore size of the primary filtration membrane is 1.0-3.0 μm, preferably 2.0 μm, and the pore size of the fine filtration membrane is less than 1.0 μm, such as 0.8, 0.6, 0.45, 0.3 μm, etc.

[0040] More preferably, the filtration step includes ultrafiltration and concentration of the clarified liquid to obtain a concentrated liquid. Further preferably, the ultrafiltration and concentration includes ultrafiltration and concentration of the monovalent virus clarified liquid using an ultrafiltration membrane of 450-550KD, preferably 500KD, respectively, concentrating the monovalent virus clarified liquid to 1 / 40-1 / 60, preferably 1 / 51 volume, and adding buffer for cyclic diafiltration. After diafiltration to the original volume of the monovalent virus clarified liquid, stop diafiltration, and concentrate to 1 / 40-1 / 60 of the original volume, preferably 1 / 51 volume, and harvest the virus concentrate. In a specific embodiment, the buffer is 0.01mol / L PBS.

[0041] More preferably, the concentrate is centrifuged after the ultrafiltration concentration step. More preferably, the centrifugation uses sucrose density gradient centrifugation to purify the monovalent virus concentrate.

[0042] More preferably, the centrifuge is desugared after the centrifugation purification step to obtain a monovalent virus gradient post-liquid. In a specific embodiment, a 450-550KD, preferably 500KD ultrafiltration membrane is selected to wash and filter the monovalent virus centrifuge to remove sugar. Wash and filter to 15-25, preferably 20 times the volume of the monovalent virus centrifuge, and then concentrate to the original volume of the monovalent virus concentrate. That is, the monovalent virus gradient post-liquid.

[0043] More preferably, the step further comprises a purification step, which may be performed after harvesting, lysis, or inactivation. Further preferably, the purification comprises chromatography purification, and in a specific embodiment, the purification step comprises chromatography purification after the desugaring step, and further preferably, a nonionic detergent, such as Tween 80, is added in a ratio of 1:(3500-4500), preferably 1:4000, for chromatography purification.

[0044] More preferably, the lysis step comprises lysing the virus solution with a lysis agent, and the protein concentration in the virus solution is 0.5-2.5 mg / ml. Further preferably, the lysis agent includes but is not limited to TritonX-100, sodium deoxycholate, and the like.

[0045] More preferably, the inactivation step includes inactivating the virus solution with an inactivator to obtain a monovalent stock solution, and the protein concentration in the virus solution is 0.5-2.5 mg / ml. Further preferably, the fire extinguishing agent includes but is not limited to formaldehyde, β-propiolactone (BPL) and the like.

[0046] More preferably, the step II) further comprises mixing the monovalent stock solutions to obtain split multivalent influenza vaccine stock solutions.

[0047] In another aspect, the present invention provides an application of the above-mentioned combined vaccine, wherein the application includes any of the following:

[0048] (A) Use in the preparation of products for preventing and / or treating diseases caused by pneumococcus and influenza virus or their complications;

[0049] (B) Use in the preparation of products for inducing immune responses to pneumococcal and influenza virus antigens;

[0050] (C) Use in the preparation of combined vaccines for use with other pathogens;

[0051] (D) Prevention and / or treatment of diseases caused by pneumococcus and influenza virus or their complications.

[0052] The beneficial effects of the present invention include:

[0053] (1) Compared with known multivalent pneumococcal vaccines, the pneumococcal vaccine of the present invention contains more common serotypes, can induce the body to produce effective immune protection, and has a broad spectrum. In particular, against serotypes 6A, 17A and 22F, the present invention can produce higher antibody titers, thereby obtaining better immune protection.

[0054] (2) After being combined with the influenza virus vaccine, there is no mutual inhibition between the two vaccine antigen components, they are well compatible and can play a synergistic role. In addition, the combined vaccine can significantly increase the antibody titer against 10A and 15B pneumoniae polysaccharides, and the average titer against influenza virus is better than that of the single quadrivalent influenza vaccine.

[0055] (3) The diseases prevented by the combination vaccine of the present invention are all seasonal respiratory diseases, especially those of children and the elderly with relatively low immunity. The combination vaccine is convenient to use, reduces the number of vaccinations for the subjects, and prevents multiple diseases at the same time, thereby reducing the risk of contracting diseases due to multiple visits to the vaccination site and the incidence of adverse reactions caused by multiple vaccinations, including adverse reactions caused by more doses of preservatives and adjuvants due to multiple vaccinations, and the physical and psychological pain caused to infants and parents by multiple injections. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels or can be prepared by known methods.

[0058] Example 1 Preparation of quadrivalent influenza virus split vaccine stock solution:

[0059] Production process: Influenza virus strain working seeds → inoculation of 9-11 day old chicken embryos → collection of allantoic fluid → monovalent virus harvest liquid after 2.0μm+0.45μm clarification treatment → 500KD ultrafiltration membrane ultrafiltration concentration → sucrose density gradient centrifugation → 500KD ultrafiltration membrane filtration → Sepharose 4FF chromatography → lysis → 30KD ultrafiltration membrane filtration → inactivation → 30KD ultrafiltration membrane filtration → 0.22μm sterilization → monovalent stock solution → semi-finished product → finished product.

[0060] 1.1 Research on vaccine stock solution production process

[0061] 1.1.1 Sources of influenza virus strains

[0062] The influenza virus strains and antisera used in this study are shown in Table 1 below:

[0063] Table 1: Influenza virus strains and antisera for detection

[0064]

[0065]

[0066] The strain was subcultured into working seed batches through SPF chicken embryos and was found to meet the quality standard requirements of the Pharmacopoeia of the People's Republic of China (2015 edition, Part III). The working seed batches of the strain were tested by the China Food and Drug Inspection Institute, and the results are as follows:

[0067] H1N1 hemagglutination titer is 1:320; virus titer is 7.1lgEID50 / ml

[0068] The hemagglutination titer of H3N2 was 1:320; the virus titer was 7.4lgEID50 / ml.

[0069] The Bv hemagglutination titer was 1:320; the virus titer was 7.7lgEID50 / ml.

[0070] By hemagglutination titer was 1:160; virus titer was 6.6lgEID50 / ml.

[0071] 1.1.2 Source of Chicken Embryos

[0072] The chicken embryos used in vaccine research and production are from healthy chickens in closed houses, 9-11 days old, without deformities, with clear blood vessels and activity. The chicken embryos come from Jilin Jiujun Animal Husbandry Co., Ltd., Jilin Guangchao Animal Husbandry Co., Ltd., Jilin Jiahong Animal Husbandry Co., Ltd., etc.

[0073] 1.1.3 Replication of influenza virus strains in chicken embryos

[0074] Referring to the "Pharmacopoeia of the People's Republic of China" (2015 edition, Volume III) Influenza Virus Split Vaccine Preparation Procedure, Item 2.3.2 Virus Inoculation and Culture Description, inoculate the appropriately diluted working seed batch virus into the allantoic cavity of chicken embryos (each type of influenza strain should be inoculated according to the requirements of the working seed batch virus dilution), place at 33-35°C, and culture for 48-72 hours.

[0075] 1.1.4 Production process research

[0076] 1.1.4.1 Clarification and Filtration of Monovalent Virus Harvest Fluid

[0077] The color of the chicken embryo allantoic fluid is slightly yellow and turbid in appearance. Therefore, we adopted a series clarification method of first coarse filtration through a filter membrane with a pore size of 2.0 μm and then fine filtration through a filter membrane with a pore size of 0.45 μm.

[0078] 1.1.4.2 Ultrafiltration and concentration of monovalent virus harvest

[0079] The monovalent virus harvest solution was ultrafiltered and concentrated using a 500KD ultrafiltration membrane. The monovalent virus harvest solution was concentrated to 1 / 51 volume, and 0.01mol / L PBS buffer was added for cyclic filtration. After filtration to the original volume of the monovalent virus harvest solution, filtration was stopped, and the virus concentrate was harvested after concentrating to 1 / 51 volume of the original volume.

[0080] 1.1.4.3 Centrifugation of concentrate

[0081] Sucrose density gradient centrifugation was used to purify the monovalent virus concentrate. The four types of monovalent virus concentrates were centrifuged at 30,000 rpm in an ultracentrifuge for 3 hours to harvest the monovalent virus centrifuge.

[0082] 1.1.4.4 Desugaring of Monovalent Virus Centrifuge Fluid

[0083] After the monovalent virus concentrate is purified by sucrose density gradient centrifugation, the sucrose concentration in the virus liquid is about 40%. We use the diafiltration method to reduce the sucrose concentration, and use a 500KD ultrafiltration membrane to diafiltration and desugar the monovalent virus centrifuge liquid. The diafiltration is carried out to 20 times the volume of the monovalent virus centrifuge liquid, and then concentrated to the original volume of the monovalent virus concentrate. This is the monovalent virus gradient liquid.

[0084] 1.1.4.5 Chromatographic purification of monovalent virus gradient liquid

[0085] Add Tween 80 (Tween 80 is a non-ionic detergent that can reduce the mutual adsorption between proteins) to the monovalent virus gradient solution at a ratio of 1:4000. Place the monovalent virus gradient solution at 20-25°C for 2-3 hours. Use Sepharose 4FF gel filtration chromatography system for chromatography (flow rate 50 cm / h).

[0086] 1.1.4.6 Process research on cracking conditions

[0087] This study uses TritonX-100 as a lysing agent for influenza virus. Confirm that the protein concentration should be within the range of 0.5-2.5 mg / ml, use Triton X-100 solution, add it to the virus purification solution at one time, make the final concentration of Triton X-100 (M:V) to 0.8%, and lyse at 20-25℃ for 2 hours. Use 40 times the volume of PBS and use a 30KD ultrafiltration membrane to wash and filter to remove the lysing agent.

[0088] 1.1.4.7 Process research on inactivation conditions

[0089] The present invention selects formaldehyde as an inactivator for influenza virus. Confirm that the protein concentration should be within the range of 0.5-2.5 mg / ml, and add formaldehyde solution. Add formaldehyde solution according to the final concentration of 180 μg / ml. Seal the inactivation solution and place it at 2-8°C, shake it manually for 10 minutes every day, and inactivate it for 5 days. Use 20 times the volume of PBS and select 30KD ultrafiltration membrane to wash and filter to remove formaldehyde.

[0090] 1.1.4.8 Unit price stock solution

[0091] The monovalent virus inactivated solution from which the lysing agent and the inactivating agent have been removed is sterilized and filtered through a filter membrane with a pore size of 0.22 μm to obtain a monovalent stock solution.

[0092] 1.1.4.9 Quadrivalent influenza split vaccine stock solution

[0093] The hemagglutinin content of each type of monovalent stock solution should reach 132μg / ml before the four monovalent stock solutions can be mixed and packaged in 0.5ml per tube. That is, the hemagglutinin content of each type of influenza virus strain should be 16.5μg.

[0094] Example 2 Design, preparation and purification of 25-valent pneumococcal polysaccharide

[0095] 4.1 Select 25 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17A, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F) of pneumococcal culture;

[0096] 4.2 Purify the capsular polysaccharides with strong antigenicity in the above various serotypes of pneumococci respectively: after inactivation of pneumococcal fermentation broth, collect the supernatant by centrifugation, concentrate by ultrafiltration, add appropriate amount (70% by volume) of pre-cooled ethanol and acetic acid for alcohol precipitation and acid precipitation respectively according to the characteristics of each pneumococcal serotype, centrifuge to remove the precipitate, add sodium acetate solution and 2 mol / L calcium chloride solution for calcium precipitation, collect the supernatant by centrifugation, wash with 15 times volume of distilled water after concentration, collect the supernatant, filter and sterilize to obtain the polyvalent refined capsular polysaccharide stock solution, and store at -20°C for future use.

[0097] The vaccine composition formula includes 25 polysaccharide types mixed in a solid-to-total ratio of 1:1, diluted with phosphate buffer to a certain concentration, and made into a 25-valent pneumococcal vaccine, wherein each type of 25-valent pneumococcal polysaccharide is 25 μg / type. The 25-valent pneumococcal group of the present application is used in the following experiments.

[0098] Example 3: Study on the immunogenicity of 25-valent pneumococcal polysaccharide vaccine

[0099] 1. Immunization protocol (negative control, positive control)

[0100] 1. Experimental Materials and Methods

[0101] Experimental materials: 6-8 week old female SD rats.

[0102] Experimental method: Fifteen 6-8 week old SD rats were randomly divided into three groups, with 5 rats in each group. The treatment method of each group was as follows:

[0103] Include

[0104] The 25-valent pneumococcal group prepared in Example 2;

[0105] The positive control group (23-valent pneumococcal polysaccharide vaccine developed by Thermo Fisher Scientific, trade name );

[0106] Negative control group (PBS group)

[0107] Table 2 Immunization schedule of 25-valent pneumococcal vaccine and positive control group

[0108] Group Immunogen Immune volume Immunization Immunization times Number of animals 1 group 25-valent pneumococcal 0.5ml im 1 5 2 groups Positive Control 0.5ml im 1 5 3 groups PBS 0.5ml im 1 5

[0109] The above three groups were injected with the vaccine into the thigh muscle on day 0.

[0110] 2. ELISA method to detect antibodies in serum

[0111] On the 21st day after immunization, rat serum was collected for ELISA analysis. The specific steps of ELISA analysis were as follows: 200 ng of the corresponding type of pneumonia polysaccharide antigen (prepared by Genoway, batch number: 20230916) was used for coating, and rat serum was used as the primary antibody for gradient dilution. The secondary antibody (Goat Anti-Rat IgG, HRP-Linked Antibody, catalog number: SA00001-15) was diluted 1:80000. The signal was read using an ELISA reader (Shanghai Kehua, catalog number: RD-SH-012), and the titer of rat serum in each group was averaged.

[0112] Table 3 Humoral immunity of 25-valent pneumococcal vaccine and positive control group

[0113]

[0114]

[0115] The results are shown in Table 3. The results show that: this product (25-valent pneumococcal vaccine) and the positive control group (the 23-valent pneumococcal vaccine that has been marketed, )) Compared with 6A and 17A, only this product produced antibody titers; the titer of 22F type of this product was significantly better than that of the listed reference product, with a p value of 0.0027, **. The titer of this product and the listed reference product for the remaining types was equivalent. In general, this product was better than the listed reference product as a whole, and could produce high levels of antibody titers against 25 types of pneumococcal polysaccharides.

[0116] Example 4 Preparation of influenza and 25-valent pneumococcal combined vaccine

[0117] A quadrivalent influenza antigen stock solution with twice the concentration was prepared according to Example 1; a 25-valent pneumococcal combination vaccine stock solution with twice the concentration was prepared according to Example 2, and then the two stock solutions were mixed in equal volumes and placed on a magnetic stirrer for low-speed stirring for 1 hour to obtain the final quadrivalent influenza and 25-valent pneumococcal combination vaccine stock solutions.

[0118] Finally, the four types of hemagglutinin, H1N1, H3N2, BV, and BY, were 16.5μg / dose / 0.5ml (a total of 66μg / dose / 0.5ml of hemagglutinin). The 25-valent polysaccharide vaccine was prepared by a 1:1 solid-to-total ratio of 25 types of polysaccharides. The buffer was phosphate buffer. The 25-valent pneumococcal vaccine antigen contained 25μg / type / 0.5ml of pneumococcal capsular polysaccharide of each type.

[0119] Flu testing methods

[0120] The antibody titer of rat serum was determined by influenza hemagglutinin. According to the national standard influenza operation method, chicken red blood cells were used for HINI, H3N2, BV, and BY experiments, and the titer was read as the last serum dilution that produced hemagglutinin inhibition.

[0121] Detection method of 25-valent pneumonia

[0122] The ELISA method was used to detect the 25-valent pneumococcal conjugate vaccine. The corresponding 25-valent pneumococcal 25 types were coated for detection. Rat serum was collected on the 21st day after the first immunization for ELISA analysis. The specific steps of ELISA analysis are as follows: 200 ng of pneumococcal polysaccharide antigen (prepared by Genove) was used for coating per well, and rat serum was used as the primary antibody for gradient dilution. Secondary antibody (Goat Anti-Rat IgG, HRP-Linked Antibody, Catalog No.: SA00001-15)

[0123] Diluted at 1:80000. The signal was read using an ELISA reader (Shanghai Kehua, catalog number: RD-SH-012).

[0124] Example 5 Immunogenicity study of influenza and 25-valent pneumococcal combined vaccine

[0125] 1. Immunogenicity study of influenza and 25-valent pneumococcal combined vaccine

[0126] Twenty SD rats aged 6-8 weeks were randomly divided into 4 groups, and the combined vaccine (quadrivalent influenza + 25-valent pneumococcus) prepared as described in Example 4 was inoculated into the rats by intramuscular injection at a dose of 0.5 ml to study the immunogenicity of the vaccine. Immunization was performed once on day 0, and the blood inhibition titer of the 21d serum against HIN1 / H3N2 / BY / BV was detected (the determination method is shown in the sop issued by the National Influenza Center: Identification of Influenza / Avian Influenza Virus by Red Blood Cell Agglutination Inhibition Test); the ELISA method was used to determine the binding antibody titer of the 21d rat serum against the 25-valent pneumococcal vaccine. The best combination vaccine prescription was screened by combining the blood inhibition titer of influenza and the binding antibody titer of the pneumococcal vaccine. The immunization scheme is shown in Table 4; the anti-influenza antibody level of the combined vaccine is shown in Table 5; the binding antibody titer of the pneumococcal vaccine of the combined vaccine is shown in Table 6:

[0127] Table 4: Influenza and 25-valent pneumococcal combined vaccine immunization schedule

[0128]

[0129]

[0130] Table 5: Anti-influenza antibody levels after immunization with influenza and 25-valent pneumococcal conjugate vaccine

[0131]

[0132] Table 6: Antibody levels against pneumococcus after immunization with influenza and 25-valent pneumococcal conjugate vaccine

[0133]

[0134]

[0135] As can be seen from Tables 5 and 6, 21 days after immunization, compared with the influenza and 25-valent pneumococcal combined vaccine (Group 3) and the quadrivalent influenza vaccine group (Group 1), the mean hemagglutinin titers of the four influenza types HINI, H3N2, BV, and BY in the combined vaccine were better than those in the single quadrivalent influenza vaccine, but there was no significant difference.

[0136] Compared with influenza and 25-valent pneumococcal vaccine (group 3) and 25-valent pneumococcal vaccine group (group 2), the combined vaccine was significantly superior to the pneumococcal vaccine alone group in terms of 10A and 15B pneumococcal polysaccharide titers, with p values ​​of 0.0068, ** and 0.0191, *, respectively. The other types of combined vaccine groups were comparable to the pneumococcal vaccine alone group.

[0137] In summary: after the quadrivalent influenza split vaccine and the 25-valent pneumococcal polysaccharide vaccine of the present invention are combined, there is no mutual inhibition between the two vaccine antigen components, they are well compatible, and can play a synergistic role. In addition, the combined vaccine can also significantly improve the antibody titer against 10A and 15B types of pneumococcal polysaccharides.

[0138] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A multivalent pneumococcal combination vaccine, characterized in that: The combined vaccine comprises: (1) a polyvalent pneumococcal capsular polysaccharide, the polysaccharide being a polysaccharide from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17A, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; (2) An influenza vaccine stock solution comprising hemagglutinin from influenza virus, wherein the influenza virus is of subtype H1N1, H3N2, B BY and BV.

2. The combined vaccine according to claim 1, characterized in that The multivalent pneumococcal capsular polysaccharide comes from a multivalent capsular polysaccharide stock solution, which includes 25 polysaccharide types, which are mixed in a solid-to-total ratio of 1:

1.

3. The combined vaccine according to claim 2, characterized in that Each type of 25-valent pneumococcal capsular polysaccharide is 10-50μg / ml.

4. The combined vaccine according to claim 1, characterized in that In the combined vaccine, the hemagglutinin content of each influenza virus subtype is 15-50 μg / ml.

5. The combined vaccine according to any one of claims 1 to 4, characterized in that: The vaccine may also include an adjuvant and / or a vaccine delivery system.

6. The method for preparing the combined vaccine according to any one of claims 1 to 5, characterized in that: The method for preparing the combined vaccine comprises: (I) a step of preparing multivalent pneumococcal capsular polysaccharide, and (II) a step of preparing influenza vaccine stock solution.

7. The preparation method according to claim 6, characterized in that: The step (I) of preparing the multivalent pneumococcal capsular polysaccharide comprises: 1) Cultivate pneumococci; 2) Purify pneumococcal capsular polysaccharide.

8. The preparation method according to claim 7, characterized in that: The step 2) comprises: 2-1) inactivating the pneumococcal fermentation broth, centrifuging and collecting the supernatant, and concentrating it by ultrafiltration; 2-2) concentrating the concentrate by alcohol precipitation, acid precipitation and calcium precipitation, and collecting the supernatant after concentration; 2-3) filtering and sterilizing to obtain a polyvalent capsular polysaccharide stock solution.

9. The preparation method according to claim 8, characterized in that: In the step 2-2), ethanol is used for alcohol precipitation, acetic acid is used for acid precipitation, and / or calcium chloride solution is used for calcium precipitation.

10. The preparation method according to claim 8 or 9, characterized in that: The multivalent capsular polysaccharide stock solution was stored at -20°C.

11. The preparation method according to claim 6, characterized in that: The preparation steps of the influenza vaccine stock solution include influenza virus amplification, harvesting, lysis and inactivation.

12. The preparation method according to claim 11, characterized in that: The preparation step also includes purification, and the purification step can be after harvesting, after lysis, or after inactivation.

13. Use of the combined vaccine according to any one of claims 1 to 5, characterized in that: The application includes any of the following: (A) Use in the preparation of products for the prevention and / or treatment of diseases caused by pneumococcus and influenza virus or their complications; (B) use in the preparation of products for inducing immune responses to pneumococcal and influenza virus antigens; (C) Application in the preparation of combined vaccines with other pathogens.

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