Pneumococcal conjugate combined vaccine and preparation method therefor

Abstract

Provided are a pneumococcal conjugate combined vaccine and a preparation method therefor. The combination comprises 26 different pneumococcal polysaccharide-protein conjugates, and the pneumococcal polysaccharide comprises a total of 26 serotypes, i.e., 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B. The carrier protein used by the conjugate is tetanus toxoid (TT). The polysaccharide is covalently linked to the TT by means of a cyano activation method so as to prepare a polysaccharide-protein conjugate stock solution, and the polysaccharide-protein conjugate stock solution is then adsorbed by an aluminum adjuvant in an appropriate proportion so as to obtain the pneumococcal conjugate vaccine. The pneumococcal polysaccharide used therein requires no degradation reaction, effectively protecting the antigenicity of the polysaccharide. Three consecutive batches of production of the conjugate stock solution and vaccine are performed, and the yield and each verification index of the conjugate are stable. The results of non-clinical safety evaluation research show that the vaccine has good safety and immunogenicity. The prepared pneumococcal conjugate vaccine is used for immunoprophylaxis of pneumococcal infection of relevant serotypes.

Description

Pneumococcal conjugate combination vaccine and preparation method thereof Technical Field

[0001] The present invention belongs to the technical field of biological product preparation, and specifically relates to a pneumococcal polysaccharide-protein conjugate combination vaccine and a preparation method thereof, as well as the use of the 26-valent pneumococcal conjugate vaccine prepared by the present invention for the immune prevention and diagnosis and treatment of pneumococcal infection. Background Art

[0002] Pneumococcal disease is a serious global public health problem. Its pathogen, the pneumococcus, colonizes the nasopharynx and typically does not cause clinical symptoms. However, when the colonization environment changes, such as when the body's immune system weakens, respiratory viral infections such as measles or influenza (flu), malnutrition, or frailty develop, pneumococci can penetrate the mucosal defense system and cause invasive infection. Approximately 75% of invasive pneumococcal disease (IPD) cases and 83% of pneumococcal meningitis occur in children under the age of two (WHO Position Paper 2019). According to the World Health Organization (WHO), more than 1.2 million people die each year from pneumococcal infection, most of whom occur in developing countries. Children under the age of two are the most vulnerable to pneumococcal disease, with a particularly high mortality rate.

[0003] Streptococcus pneumoniae secretes a capsule surrounding its cell wall, which is its primary virulence factor. Pneumococci are classified into nearly 100 serotypes based on their capsule structure, and the distribution of predominant serotypes varies across time and region.

[0004] Pneumococcal vaccines are the most effective means of preventing pneumococcal infection. Currently available vaccines are the pneumococcal polysaccharide vaccine (PPV) and the pneumococcal conjugate vaccine (PCV). In the 1980s, the 23-valent pneumococcal polysaccharide vaccine (PPV23), produced by Wyeth (acquired by Pfizer in 2009), achieved approximately 90% coverage against the dominant serotype in all continents. However, pneumococcal capsular polysaccharide is a thymus-independent (TI) antigen and does not induce an effective immune response in children under 2 years of age, an age group at high risk for pneumococcal infection. The pneumococcal polysaccharide-protein conjugate vaccine can be administered starting at 2 months of age for a full course of 3 to 4 doses. This vaccine promotes a strong immune response and memory response, making it a beneficial immunization method for faster and earlier prevention of pneumococcal infection. Studies have shown that conjugate vaccines can be used in all age groups and offer longer-lasting effects than polysaccharide vaccines. Therefore, the trend in pneumococcal vaccines is for conjugate vaccines to replace polysaccharide vaccines. The first commercially available PCV was PCV7 (Prevnar 7) produced by Wyeth, which received FDA approval in 2000. In 2010, PCV13 (Prevnar 13), which covers a wider range of vaccine types, was released, replacing Prevnar 7 for routine childhood vaccination. Currently, three PCVs have received WHO prequalification: PCV10 (Synflorix) from GlaxoSmithKline, PCV13 (Prevenar 13) from Pfizer, and PCV10 (PNEUMOSIL) from the Serum Institute of India. In 2021, the FDA approved two higher-priced PCVs: Pfizer's Prevnar 20 (PCV20) and Merck's VAXNEUVANCE (PCV15). In China, two pneumococcal polysaccharide conjugate vaccines are available: Watson Biopharma's PCV13 and Minhai Biopharma's PCV13.

[0005] The currently available pneumococcal polysaccharide protein conjugate vaccine is a chemical method that covalently binds the polysaccharide antigen to the carrier protein. There are two main methods: amine reduction method and cyano group activation method. (1) Reduction amine method. The polysaccharide is oxidized with sodium periodate, and the aldehyde or ketone group formed reacts with the active amino group of the carrier protein to form a C=N double bond, which is reduced to a CN single bond by a reducing agent to form a stable covalent bond. (2) Cyano group activation method. A cyano group is introduced into the polysaccharide by cyanogen bromide (CNBr) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), and then forms a covalent bond with the protein primary-NH2. With the development of conjugation technology, based on these two conjugation methods, some new conjugation methods have been developed, such as introducing a molecular arm adipic acid dihydrazide (ADH) between the polysaccharide and the protein to promote the binding of the polysaccharide and the protein, or introducing a new linking group to promote the occurrence of the conjugation reaction.

[0006] However, literature reports suggest that prior to the polysaccharide-protein binding reaction, the molecular size of the polysaccharide must be reduced to a certain extent. Common degradation methods include physical degradation, chemical degradation, and enzymatic degradation. Physical degradation generally includes microwaves, radiation, ultrasonic degradation, and high-pressure homogenizer shearing. Chemical degradation includes acid hydrolysis, alkaline hydrolysis, and hydrogen peroxide degradation. Enzymatic degradation utilizes specific or non-specific enzymes to degrade polysaccharides.

[0007] CN108079286A "A 13-valent pneumococcal polysaccharide-protein conjugate composition and its preparation method and application" discloses a method for detecting K on a Sepharose CL-4B column for molecular size. D Pneumococcal capsular polysaccharide with a K value < 0.20 was subjected to ultrasonic degradation or high temperature hydrolysis at 70-85°C before activation and derivatization to reduce the molecular size of the polysaccharide to a level that can be detected on a Sepharose CL-4B column. D The value is between 0.20-0.50.

[0008] CN109862908B “Multivalent pneumococcal polysaccharide-protein conjugate composition” discloses a method for hydrolysis using sodium hydroxide, hydrochloric acid and glacial acetic acid under high temperature conditions.

[0009] CN107810010A “Multivalent Pneumococcal Conjugate Vaccine” discloses the use of a high-pressure homogenizer to reduce the molecular size of polysaccharides.

[0010] CN103830723A “A method for preparing a pneumococcal capsular polysaccharide protein conjugate vaccine” discloses that a polysaccharide solution is treated with ultrasound at a power of 80-100W and a frequency of 20-40kHz in an ice-water bath.

[0011] Polysaccharide antigenicity is a key indicator of immune reactivity and immunogenicity. The larger the relative molecular mass of a polysaccharide, the stronger its antigenicity. A decrease in relative molecular mass may be accompanied by a loss of antigenicity. It has been shown that each polysaccharide degradation method, while reducing the molecular size of the polysaccharide, also destroys its specific groups and antigenic epitopes, leading to significant batch-to-batch variability in the quality attributes of the degraded polysaccharide. Summary of the Invention

[0012] The present invention overcomes the aforementioned problem of polysaccharide antigenicity loss. It utilizes refined polysaccharides harvested through bacterial fermentation and polysaccharide purification. In the polysaccharide-protein conjugation process, the refined polysaccharide undergoes no degradation treatment, directly undergoing polysaccharide activation and conjugation reactions. This use of naturally purified pneumococcal polysaccharide effectively preserves the polysaccharide's antigenicity. Three consecutive batches of conjugate stock solutions and vaccines were produced, and the polysaccharide yield and various assay parameters were stable, confirming the stability of the present process.

[0013] In one aspect, the present invention provides a 26-valent pneumococcal conjugate combination vaccine.

[0014] The 26-valent pneumococcal conjugate combination vaccine of the present invention is based on the capsular polysaccharide of the pneumococcus and covers the most common serotypes that cause pneumococcal disease. The present invention adds three serotypes, 6A, 24F, and 35B, to the original 23-valent pneumococcal polysaccharide vaccine (serotypes include 1, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, a total of 23). Among them, type 6A is the serotype in the 13-valent pneumococcal conjugate vaccine PCV13, and serotypes 24F and 35B are two serotypes with a relatively significant increase in invasive pneumococcal disease caused by non-vaccine serotypes. According to literature reports, serotype 24F has received widespread attention in recent years and is particularly common in cases of drug-resistant invasive pneumococcal disease. Countries including Germany, the United Kingdom, Spain, Denmark, and Japan have reported an annual increase in cases of invasive pneumococcal disease caused by serotype 24F. Invasive pneumococcal disease caused by serotype 35B has also seen a significant increase in recent years, with reports in countries including China, the United States, and South Africa. While the 23 original serotypes provide approximately 90% immunization coverage across all continents, the present invention, with the addition of three new serotypes, expands the scope of immune protection to well over 90%.

[0015] A technical solution of the present invention is as follows: a 26-valent pneumococcal conjugate combination vaccine comprises 26 pneumococcal serotypes, wherein the conjugate is a polysaccharide-protein conjugate prepared by chemical covalent bonding of pneumococcal capsular polysaccharides of corresponding serotypes with a carrier protein, tetanus toxoid; the 26 polysaccharide-protein conjugate stock solutions are then adsorbed with an aluminum adjuvant in appropriate proportions to obtain the pneumococcal conjugate vaccine.

[0016] In one embodiment, the 26-valent pneumococcal conjugate combination vaccine of the present invention further comprises a pharmaceutically acceptable excipient or carrier.

[0017] In one embodiment, the 26-valent pneumococcal conjugate combination vaccine of the present invention includes 26 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B.

[0018] Based on the 23-valent pneumococcal polysaccharide vaccine, three serotypes, 6A, 24F and 35B, were added, and the immunization coverage rate far exceeded 90%.

[0019] Among them, the pneumococcal capsular polysaccharides except those of types 2 and 3 were activated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP), and then adipic dihydrazide (ADH) was used as a spacer to form derivatives, which were then reacted with the carrier protein TT under the action of carbodiimide (EDAC) to form polysaccharide-protein conjugates.

[0020] Among them, type 2 and type 3 polysaccharides were activated by CDAP and then directly combined with TT to form polysaccharide-protein conjugates.

[0021] In one embodiment, the vaccine further comprises an adjuvant. In one embodiment, the adjuvant is an aluminum adjuvant. In one embodiment, the aluminum adjuvant is an aluminum phosphate adjuvant.

[0022] In another aspect, the present invention provides a method for preparing a 26-valent pneumococcal conjugate combination vaccine.

[0023] In the preparation method of the 26-valent pneumococcal conjugate combination vaccine of the present invention, pneumococcal polysaccharide and TT are covalently bound by a cyano activation method to prepare a polysaccharide-protein conjugate. Type 2 and type 3 polysaccharides are directly bound by the direct binding method, that is, the polysaccharide is activated by CDAP and then directly bound to TT to form a polysaccharide-protein conjugate. The polysaccharides of other serotypes of pneumococcus are first derivatized and then bound by the binding method, that is, the polysaccharide is activated by CDAP and then adipic dihydrazide (ADH) is used as a spacer to form a derivative, which is then bound to the carrier protein TT under the action of carbodiimide (EDAC) to form a polysaccharide-protein conjugate. In one embodiment, the preparation method is as follows:

[0024] Step 1: After the pneumococcal polysaccharide is fermented by the strain and the bacterial cells are removed, the polysaccharide is purified by CTAB precipitation, sodium chloride dissociation, hydroxyapatite chromatography, and finally freeze-drying to obtain refined pneumococcal polysaccharide;

[0025] Step 2: Activating pneumococcal polysaccharides of types 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F, and 35B with CDAP, derivatizing with ADH as a spacer, and purifying by ultrafiltration to form polysaccharide derivatives, which are then condensed with carrier protein TT under the action of EDAC, and ultrafiltration to obtain polysaccharide-protein conjugates;

[0026] The CDAP:polysaccharide mass ratio is 0.1-1.0, preferably 0.1-0.5 for 8, 9N, 9V, 10A, 20, 33F and 35B, and 0.5-1.0 for other types;

[0027] Among them, the final concentration of ADH was 0.2 mol / L;

[0028] The reaction process has a pH of 8.0 to 9.0;

[0029] The reaction time is no less than 2 hours.

[0030] Step 3: Type 2 and type 3 polysaccharides were activated by CDAP and then directly reacted with TT, and polysaccharide-protein conjugates were obtained by ultrafiltration;

[0031] wherein the CDAP:polysaccharide mass ratio is 0.1 to 1.0, preferably 0.25 to 0.75;

[0032] Wherein, the mass ratio of polysaccharide to carrier protein TT is 1:0.5 to 1:2, preferably 1:1 to 1:2;

[0033] Among them, the reaction process pH is 8.0-9.0,

[0034] The reaction time is no less than 2 hours.

[0035] Step 4: The polysaccharide-protein conjugate was chromatographed on Sepharose 4FF, and the fractions near V0 and K D The components before 0.2 are the purified conjugates, and after sterilization and filtration, they are the pneumococcal polysaccharide protein conjugate stock solution;

[0036] Step 5: Mix the 26 types of polysaccharide-protein conjugate stock solutions and add Tween 80, aluminum phosphate adjuvant and sodium chloride solution. After filling, the 26-valent pneumococcal conjugate combination vaccine is obtained.

[0037] In one embodiment, the weight-average molecular weight (Mw) of most types of purified pneumococcal polysaccharide is between 300 and 800 kDa, with types 2 and 7F having Mws between 900 and 1200 kDa, and types 18C, 19A, and 19F having Mws between 100 and 400 kDa. Except for types 6A, 24F, and 35B, the quality of the other 23 types of purified polysaccharides meets the polysaccharide quality standards for "23-valent pneumococcal polysaccharide vaccines" in the 2020 Pharmacopoeia of the People's Republic of China. The quality of purified polysaccharides of types 6A, 24F, and 35B meets the approved requirements.

[0038] In one embodiment, the derivatization rate (ADH content) of the pneumococcal polysaccharide derivatives of types 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F, and 35B is 1% to 10%. The ADH content is controlled by controlling the amount of CDAP added during the derivatization reaction and other reaction parameters, thereby avoiding excessive cross-linking of the polysaccharide-protein conjugate and ensuring smooth chromatographic purification and sterile filtration.

[0039] The method for preparing the 26-valent pneumococcal conjugate combination vaccine of the present invention may further comprise: weighing 26 polysaccharide-protein conjugate stock solutions, adding them to a Tween 80 solution, and stirring uniformly; mixing the mixed stock solution with an aluminum phosphate adjuvant, and adsorbing the mixture overnight; and dispensing the semi-finished product into sterile 1 ml prefilled syringes, which are then filled to obtain the finished product.

[0040] In one embodiment, the pH of the vaccine is 5.0-7.0, the aluminum ion content is 0.15-0.35 mg / ml, the sodium ion content is 7.5-9.5 g / L, and the Tween 80 content is 120-180 μg / ml.

[0041] In one embodiment, in a unit dose of the combination vaccine, the content of each type of pneumonia polysaccharide is 2.2 μg±30%, and that of 6B is 4.4 μg±30%, and the above unit dose is 0.5 ml.

[0042] In one embodiment, a stock solution of type 2 and type 3 pneumococcal polysaccharide protein conjugate is prepared as follows:

[0043] 1 g of polysaccharide was weighed and activated by adding 100 mg / ml CDAP acetonitrile solution (CDAP:polysaccharide mass ratio was 0.10-0.75). A triethylamine aqueous solution was added to maintain the pH at 8.0-9.0. A carrier protein was added (polysaccharide:protein feed ratio was 1:1-1:2), and the pH was maintained at 8.0-9.0. The reaction time was not less than 2 hours, and a polysaccharide-protein conjugate was obtained by ultrafiltration or dialysis.

[0044] In one embodiment, a stock solution of type 2 and type 3 pneumococcal polysaccharide protein conjugate is prepared as follows:

[0045] Preparation of stock solutions of 24 types of pneumococcal polysaccharide-protein conjugates except types 2 and 3

[0046] 1 g of polysaccharide was weighed and activated by adding 100 mg / ml CDAP acetonitrile solution (CDAP:polysaccharide mass ratio of 0.10-0.75). Then, triethylamine aqueous solution was added to adjust the pH to 8.0-9.5 and maintained in the range. An equal volume of ADH solution was added to a final concentration of 0.2 mol / L and the pH was maintained in the range of 8.0-9.0. The reaction was continued for at least 2 hours. The polysaccharide derivative was purified by ultrafiltration. The polysaccharide derivative was mixed with a carrier protein and EDAC solution was added to a final concentration of 0.02 mol / L. The pH was maintained at approximately 5.6. The reaction was continued for at least 2 hours. The polysaccharide-protein conjugate was purified by ultrafiltration.

[0047] In one embodiment, a 26-valent pneumococcal conjugate vaccine is prepared as follows:

[0048] According to the content of each type of pneumonia polysaccharide in each dose of vaccine being 2.2μg±30% and 6B being 4.4μg±30%, 26 polysaccharide-protein conjugate stock solutions were weighed separately and added to Tween 80 solution and stirred evenly; the mixed stock solution was mixed with aluminum phosphate adjuvant and adsorbed overnight; the aluminum ion content was 0.15-0.35mg / ml, the sodium ion content was 7.5-9.5g / L, and the Tween 80 content was 120-180μg / ml.

[0049] The above semi-finished products are divided into sterile 1ml pre-filled syringes, and the finished products are obtained after filling. Each dose of vaccine contains 0.5ml.

[0050] In another aspect, the present invention provides use of the vaccine of the present invention in preparing a medicament for preventing or treating pneumonia.

[0051] In another aspect, the present invention provides a vaccine of the present invention for use in medicine.

[0052] In another aspect, the present invention provides the vaccine of the present invention for use in prevention or treatment.

[0053] In another aspect, the present invention provides the vaccine of the present invention for use in preventing or treating pneumonia.

[0054] In another aspect, the present invention provides a method for preventing or treating pneumonia, comprising administering to a subject a preventively or therapeutically effective amount of the vaccine of the present invention.

[0055] In one embodiment, the pneumonia is pneumonia associated with pneumococcal infection.

[0056] The vaccine of the present invention can be used to protect or treat subjects susceptible to infection by administering the vaccine through a systemic or mucosal route. These administrations can include injection via intramuscular (IM), intraperitoneal (IP), intradermal (ID) or subcutaneous (SC) routes; or administration via the mucosa of the oral / digestive tract, respiratory tract, or genitourinary tract. Although the vaccine of the present invention can be administered as a single dose, its components can also be co-administered at the same time or at different times. In addition to a single route of administration, two or more different routes of administration can be used. After the initial vaccination, the individual can receive one or several booster immunizations appropriately spaced apart.

[0057] Vaccines of the present invention can be stored in solution or freeze-dried. In one embodiment, the solution is freeze-dried in the presence of a sugar such as sucrose or lactose. It is further preferred that they are freeze-dried and temporarily reconstituted before use.

[0058] In one aspect of the present invention, a kit is provided, comprising a vial containing the vaccine of the present invention (optionally in freeze-dried form) and a vial containing an adjuvant as described herein. In this aspect of the invention, the adjuvant can be used to reconstitute the freeze-dried immunogenic composition. Preclinical animal studies of the 26-valent pneumococcal conjugate combination vaccine of the present invention have shown good safety and immunogenicity. Compared with the marketed 13-valent pneumococcal conjugate vaccine, all 26 serotypes can stimulate mice to produce high levels of antibody titers.

[0059] Compared with existing pneumococcal conjugate combination vaccines, the present invention has the following beneficial effects:

[0060] (1) The purified polysaccharide used in the polysaccharide-protein conjugate process of the 26-valent pneumococcal conjugate combination vaccine of the present invention is not subjected to any degradation treatment, and the polysaccharide activation and conjugation reaction are directly carried out. This use of naturally purified pneumococcal polysaccharide effectively preserves the antigenicity of the polysaccharide. The present invention produced three consecutive batches of conjugate stock solution and vaccine, and the polysaccharide yield and various test indicators were stable, confirming the stability of the process of the present invention.

[0061] (2) By controlling the amount of CDAP added during the derivatization reaction and other reaction parameters, the derivatization rate was controlled, thus avoiding excessive cross-linking of the polysaccharide-protein conjugate and ensuring smooth chromatographic purification and sterilization filtration.

[0062] (3) The antigenicity of pneumococcal polysaccharide, conjugates, and intermediates in their preparation was monitored and analyzed using rate turbidimetry, providing a reference for the selection of pneumococcal polysaccharide protein conjugation process routes. This minimized the destruction of the specific epitopes of the antigens in the vaccine stock solution and protected the integrity of the antigens.

[0063] (4) The results of preclinical animal experiments showed that the 26-valent pneumococcal conjugate combination vaccine of the present invention has good safety and immunogenicity. Compared with the 13-valent pneumococcal conjugate vaccine already on the market, the antibody titers of 13 serotypes were not inferior to the antibody titers of the same serotypes in the 13-valent pneumococcal conjugate vaccine. The other 13 serotypes were also able to stimulate mice to produce high levels of antibodies. DETAILED DESCRIPTION

[0064] Further explanation of the English abbreviations or technical terms appearing in the manual:

[0065] "Polysaccharide" refers to a complex carbohydrate composed of chains of sugars linked together by glycosidic bonds. A polysaccharide may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more sugars. The polysaccharide of the present invention refers to the capsular polysaccharide of Streptococcus pneumoniae.

[0066] The "molecular weight" of a polysaccharide refers to the weight average molecular weight (Mw) of the polysaccharide as measured by size exclusion chromatography (SEC) or multi-angle laser light scattering (MALLS) prior to conjugation.

[0067] "Conjugate" refers to the product obtained by covalently or non-covalently linking pneumococcal capsular polysaccharide and a carrier protein.

[0068] Carrier protein: Common carrier proteins in the art can be selected from TT (tetanus toxoid), DT (diphtheria toxoid), CRM197, fragment C of TT, PhtD (pneumococcal histone triad protein D), PhtDE fusion (fusion of PhtD and PhtE (pneumococcal histidine protein E), detoxified pneumolysin and protein D, etc. The TT used in the present invention is prepared by inoculating Clostridium tetani with strong toxin production in a suitable culture medium, and inactivating and detoxifying the produced exotoxin with formaldehyde solution, followed by sterilization and filtration. It is commonly used as a carrier protein in conjugate vaccines.

[0069] "26-valent pneumococcal conjugate combination vaccine" refers to a composition comprising pneumococcal capsular polysaccharide-protein conjugate, wherein the pneumococcal capsular polysaccharide-protein conjugate comprises or consists of 26 different pneumococcal capsular polysaccharide-protein conjugates, wherein each pneumococcal capsular polysaccharide-protein conjugate comprises a carrier protein conjugated to a capsular polysaccharide from a different serotype of Streptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypes include 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B.

[0070] "Human dose" means a dose in a volume suitable for human use. Typically, this is 0.25-1.5 ml. In one embodiment, the human dose is 0.5 ml. In further embodiments, the human dose is greater than 0.5 ml, such as 0.6, 0.7, 0.8, 0.9, or 1 ml. In further embodiments, the human dose is 1 ml-1.5 ml. In another embodiment, particularly when the immunogenic composition is used in a pediatric population, the human dose may be less than 0.5 ml, such as 0.25-0.5 ml.

[0071] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers that can be used in the present invention are conventional. Suitable pharmaceutical excipients or carriers include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. Typically, the nature of the excipient or carrier will depend on the specific mode of administration used. For example, parenteral formulations typically include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, normal saline, balanced salt solutions, buffers, dextrose solutions, glycerol, etc. as vehicles. For solid compositions (e.g., powder, pill, tablet, or capsule form), conventional non-toxic solid excipients can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the vaccine to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, surfactants, preservatives and pH buffers, for example sodium acetate or sorbitan monolaurate.

[0072] "Adjuvant" is a non-specific immunoproliferative agent, which refers to auxiliary substances that can enhance the body's immune response to antigens or change the type of immune response when injected into the body together with the antigen or in advance. Adjuvants can be immunogenic or non-immunogenic. The immunobiological effects of adjuvants are to enhance immunogenicity, enhance the titer of antibodies, change the type of antibody produced, and cause or enhance delayed hypersensitivity reactions. Adjuvants can include: bacteria or their products, which are immunogenic in themselves, such as mycobacteria (tuberculosis, BCG), Corynebacterium brevis, Bordetella pertussis, Gram-negative bacilli endotoxin, etc.; inorganic adjuvants, such as aluminum hydroxide, alum, aluminum phosphate, etc.; synthetic adjuvants, such as double-stranded polyinosinic acid, cytidylic acid, double-stranded polyadenylic acid, etc.; oils, such as peanut oil emulsified adjuvant, mineral oil, vegetable oil, lanolin, etc.; Freund's adjuvant. The present invention can use aluminum adjuvants, but is not limited to them.

[0073] A "prophylactically or therapeutically effective amount" refers to an amount or dosage required to induce an immune response sufficient to delay the onset and / or reduce the frequency and / or severity of one or more symptoms caused by S. pneumoniae infection.

[0074] "Subject" means any mammal, including humans. In some embodiments, the subject is an elderly person, an adult, a teenager, or an infant.

[0075] CTAB: hexadecyltrimethylammoniumbromide, hexadecyltrimethylammonium bromide, is a cationic detergent that can be used to precipitate polysaccharides.

[0076] CDAP: 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, CDAP can be used to activate polysaccharides.

[0077] ADH: Adipoyl Hydrazide, adipic acid dihydrazide, also known as adipic dihydrazide. ADH is an excellent coupling agent that can chemically cross-link with aldehyde groups.

[0078] EDAC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, carbodiimide, used as a carboxyl activation reagent in amide synthesis.

[0079] Sepharose 4FF: Sepharose 4Fast Flow, a gel filtration chromatography filler. It is a liquid chromatography method that uses a porous gel filler as the stationary phase and separates components by molecular size.

[0080] V0: The external volume refers to the volume of the liquid mobile phase between the gel particles in the chromatography column. Since macromolecules do not enter the interior of the gel particles but only exist in the mobile phase between the gel particles, their elution volume is equal to V0. Polysaccharide-protein conjugates are such macromolecules, so the elution peak near V0 is collected in gel chromatography.

[0081] Hydroxyapatite (CHT): Ceramic hydroxyapatite is a hydroxy compound composite mode chromatography medium with important application value. It has a spherical appearance and ultra-large pores. It is divided into two types: CHT I and CHT II.

[0082] The present invention is further illustrated by the following examples.

[0083] The product and its preparation method of the present invention are further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified.

[0084] The raw materials used in the examples can be purchased on the market or prepared according to existing methods.

[0085] The polysaccharide and carrier protein TT were prepared by our company according to existing methods. The bacterial strains used were purchased from the China Medical Bacteria Collection Center of the China Food and Drug Administration. The main chemical agents were purchased from Sigma.

[0086] Example 1: Preparation and Assay Results of Pneumococcal Polysaccharide

[0087] Twenty-six pneumococcal serotype strains were obtained from the China Medical Bacteria Collection Center of the China Food and Drug Administration. These strains were subcultured to establish a library and prepare a working seed batch. From the time the working seed batch was opened until inoculation into the fermenter, the number of subcultures should not exceed five. Cultivation was terminated in the late logarithmic growth phase or early stationary phase, and sodium deoxycholate was added for sterilization.

[0088] The sterilized culture fluid is centrifuged and the supernatant is collected. The supernatant is precipitated with 1% to 3% CDAB, purified by ultrafiltration, purified by hydroxyapatite (CHT II) chromatography, the flow-through peak is collected, concentrated by ultrafiltration, and freeze-dried to obtain the purified pneumococcal polysaccharide.

[0089] The quality of the refined polysaccharides from 23 types, except for 6A, 24F, and 35B, meets the polysaccharide quality standards for 23-valent pneumococcal polysaccharide vaccines in the 2020 Pharmacopoeia of the People's Republic of China. The quality of the refined polysaccharides from 6A, 24F, and 35B types also meets the requirements. The test results for the 23 serotypes of pneumococcal polysaccharides are shown in the table below.

[0090] Table 1 Test results of 23 serotypes of pneumococcal polysaccharides

[0091] Example 2: Preparation and Assay Results of Type 2 and Type 3 Pneumococcal Polysaccharide Protein Conjugates

[0092] Weigh 1g of polysaccharide and add 100mg / ml CDAP acetonitrile solution (CDAP: polysaccharide mass ratio is 0.10-0.75) for activation. At the same time, add triethylamine aqueous solution to maintain pH 8.0-9.0. Add carrier protein (polysaccharide: protein weight ratio is 1:1-1:2) and maintain pH 8.0-9.0. The reaction time is not less than 2 hours. The polysaccharide-protein conjugate is obtained by ultrafiltration or dialysis. The polysaccharide-protein conjugate is collected by Sepharose 4FF column chromatography near V0, K D The components before 0.2 are the purified conjugates, and after being sterilized and filtered through 0.22 μm, they are the stock solution of the conjugate.

[0093] Table 2 Test results of type 2 and type 3 pneumococcal polysaccharide protein conjugate stock solution

[0094] Example 3: Preparation and Assay Results of 24 Pneumococcal Polysaccharide-Protein Conjugates in Excluding Types 2 and 3

[0095] Weigh 1g of polysaccharide, add 100mg / ml CDAP acetonitrile solution (CDAP: polysaccharide mass ratio is 0.10-0.75) for activation, add triethylamine aqueous solution to adjust to pH 8.0-9.5, maintain the pH range of 8.0-9.5, add an equal volume of ADH solution to a final concentration of 0.2mol / L, maintain the pH range of 8.0-9.0, react for no less than 2 hours, and obtain polysaccharide derivatives after ultrafiltration purification. The polysaccharide derivatives are mixed with the carrier protein, and EDAC solution is added to a final concentration of 0.02mol / L, maintain the pH at around 5.6, react for no less than 2 hours, and obtain polysaccharide-protein conjugates by ultrafiltration purification. The conjugates are purified by Sepharose 4FF column chromatography, with a V0 of around 0.02mol / L and a K of 0.02mol / L. D The fractions before 0.2 μm are the purified conjugates, and after sterilization and filtration through 0.22 μm, they are the stock solution of the conjugate. The results of the derivatization yield test for three consecutive batches of 24-type pneumococcal polysaccharide derivatives are shown in Table 3, and the results of the stock solution of 24-type pneumococcal polysaccharide-protein conjugates are shown in Table 4.

[0096] Table 3 Results of the derivatization rate test of type 24 pneumococcal polysaccharide derivatives

[0097] Table 4 Test results of 24-type pneumococcal polysaccharide protein conjugate stock solution

[0098] Example 4: Preparation of 26-valent pneumococcal conjugate vaccine

[0099] To ensure that each dose of vaccine contains 2.2 μg ± 30% of each pneumonia polysaccharide (except for 6B, which contains 4.4 μg ± 30%), weigh out the stock solutions of 26 polysaccharide-protein conjugates and add them to a Tween 80 solution, stirring evenly. The mixed stock solution is then adsorbed with aluminum phosphate adjuvant overnight. The aluminum ion content is 0.15-0.35 mg / ml, the sodium ion content is 7.5-9.5 g / L, and the Tween 80 content is 120-180 μg / ml.

[0100] The semi-finished product is divided into sterile 1ml pre-filled syringes and the finished product is obtained after filling. Each dose of vaccine contains 0.5ml.

[0101] The specific quantitative test was performed to determine the content of each type in the finished product. The results are shown in Table 5.

[0102] Table 5 Finished product test results

[0103] Example 5: Study on the relative antigenicity of polysaccharides and degraded polysaccharides

[0104] In previous studies, we conducted extensive research on the relative antigenicity of type 26 polysaccharides and degraded polysaccharides using rate turbidimetry, and conducted acid hydrolysis and high-pressure homogenizer shear tests. (1) Taking type 4 as an example, the hydrolysis reaction was carried out at 40°C and a final concentration of 0.5 mol / L glacial acetic acid. The antigen of the purified polysaccharide was set to 100, and the relative antigenicity of the degraded polysaccharide was measured. The results are shown in Table 6. (2) Taking types 10A and 33F as examples, the high-pressure homogenizer pressure was set to 1000 Bar, and shearing was performed 30 times. Samples were taken at 2, 4, 6, 8, 10, 20, and 30 times to measure the relative antigenicity of the degraded polysaccharide. The results are shown in Table 7.

[0105] The results of the relative antigenicity comparison test showed that both acid hydrolysis and high-pressure homogenizer shearing reduced the antigenicity of the polysaccharide to varying degrees. Therefore, the polysaccharide selected in the present invention was not degraded by any method.

[0106] Table 6 Results of relative antigenicity studies under acid hydrolysis conditions for type 4

[0107] Table 7 Relative antigenicity of type 8 and type 33F under high pressure homogenization conditions

[0108] Example 6: Study of relative antigenicity of type 2 and type 3 in different binding methods

[0109] The immunoreactivity retention (i.e., antigenicity) of samples from the preparation of pneumococcal polysaccharides and their polysaccharide conjugates was determined using rate turbidimetry. The immunoreactivity retention of the polysaccharide was set to 100, and the relative retention of immunoreactivity for samples of the same concentration (e.g., derivatives, conjugate stock solutions) was calculated.

[0110] The results of rate turbidimetry showed that the immunoreactivity retention of type 2 and type 3 pneumococcal polysaccharide conjugates was poor, especially the type 3 rate turbidimetry result was almost 0.

[0111] Subsequently, by changing the binding process, the direct binding method was used to prepare type 2 and type 3 polysaccharide protein conjugate stock solutions. The rate turbidimetric results showed that the immunoreactivity of the conjugate stock solutions was well retained. Two batches of each type and each method were selected for rate turbidimetric determination and the results were compared, see Table 8. Therefore, the direct binding method was finally used for type 2 and type 3.

[0112] Table 8 Results of relative antigenicity studies of different binding methods for type 2 and type 3

[0113] Example 7: Investigation of process stability

[0114] The present invention produced three consecutive batches of conjugate stock solutions and vaccines. The polysaccharide derivative derivatization rates for these three batches are shown in Table 3. The polysaccharide derivatization rates for each serotype were not significantly different. Comprehensive analytical testing of the three consecutive batches of stock solutions demonstrated compliance with the established quality standards. The total yields of the three batches were consistent, with good inter-batch consistency. The free polysaccharide and polysaccharide yields of the three consecutive stock solutions, using serotypes 9N, 9V, 10A, and 11A as examples, are shown. All three consecutive vaccine batches met the quality standards, demonstrating the stability of the present invention's process.

[0115] Table 9 Results of free polysaccharide and polysaccharide yield of 9N, 9V, 10A and 11A stock solutions

[0116] Example 8: Comparison of immunogenicity with the 13-valent pneumococcal conjugate vaccine (cyano-activated method) already available in China

[0117] Animal trials were conducted using a similar domestically marketed product, the 13-valent pneumococcal conjugate vaccine, as a control to compare immunogenicity. The immunization protocol employed was as follows: 1 / 2 dose of each vaccine was administered subcutaneously to NIH mice, once every two weeks for three consecutive doses.

[0118] Experimental method: The experiment used 30 female NIH mice, weighing 12-16g, and randomly divided them into 3 groups, namely negative control group, 26-valent pneumococcal conjugate vaccine group, and 13-valent pneumococcal polysaccharide conjugate vaccine group, with 10 mice in each group. The negative control group was given 0.85% sodium chloride injection; the experimental groups were given 26-valent pneumococcal conjugate vaccine and 1 / 2 dose of the marketed 13-valent pneumococcal polysaccharide conjugate vaccine respectively. When the animals were administered, they were diluted according to a volume of 0.5ml per dose and then administered. The control vaccine or the test sample was diluted 2 times and administered at a volume of 0.5ml per mouse. The serum collected 14 days after each administration (i.e., one-, two-, and three-immunity sera) was tested for serum-specific antibody titer and positive conversion rate. The results are shown in Table 10.

[0119] As can be seen from Table 10, the 26-valent pneumococcal conjugate vaccine is compared with the domestic 13-valent pneumococcal conjugate vaccine. For the 13 common types (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), the geometric mean titers (GMTs) of antibodies for types 1, 7F, and 14 in the triple-immune serum are slightly lower than those of the 13-valent pneumococcal conjugate vaccine that has been marketed, but the geometric mean titers of antibodies are all within 3 times the range; the remaining 10 types are not lower than those of the marketed vaccine; for the 13 types unique to the 26-valent vaccine (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 24F, 33F, and 35B), all can stimulate mice to produce high-level antibody titers.

[0120] Table 10 Comparison of immunogenicity with 13-valent pneumococcal conjugate vaccines available in China

[0121] Example 9 Immunogenicity Test in Sprague-Dawley (SD) Rats

[0122] Experimental Methods: 30 SD rats were randomly divided into three groups, half male and half female. The negative control group was given 1.5 ml of sodium chloride injection per rat. The low- and high-dose groups were given 26-valent pneumococcal conjugate vaccine intramuscularly, with a dose of 1 and 3 doses per rat, respectively, in volumes of 0.5 ml and 1.5 ml per rat. The vaccine was administered every two weeks for eight consecutive weeks, as well as during the two-week and four-week recovery periods (D14, D28, D42, D56, D70, and D84) for serum antibody titer testing.

[0123] The results are shown in Table 11. The 26-valent pneumococcal conjugate vaccine had good immunogenicity in rats. The sera of the immunized rats produced high levels of antibody titers, and the high antibody levels were maintained during the recovery period.

[0124] Table 11 Summary of specific IgG antibodies in the serum of animals in each drug-treated group

[0125] Example 10: Comparison of immunogenicity of monovalent vaccine and 26-valent pneumococcal conjugate vaccine

[0126] The test method was the same as in Example 8, except for the negative control group and the test group. The 26 serotype polysaccharide-protein conjugate stock solutions were prepared into monovalent vaccines (without adjuvant) and 26-valent pneumococcal conjugate vaccines (without adjuvant) according to the finished product formulations. Animal experiments were conducted to compare the immunogenicity. The immunization procedure employed was as follows: the two groups of vaccines were administered to NIH mice (subcutaneously) at a human dose per mouse, once every two weeks for three consecutive doses. Serum-specific antibody titers were measured in serum collected 14 days after each administration (i.e., one-, two-, and three-immune sera). The results are shown in Table 12.

[0127] As can be seen from Table 12, the geometric mean titer of antibodies in the 26-valent pneumococcal conjugate vaccine compared with the monovalent pneumococcal conjugate vaccine, among which the geometric mean titer of antibodies in the 3, 4, 7F and 14 type triple immune sera was slightly lower than that in the monovalent pneumococcal conjugate vaccine, the geometric mean titer of antibodies in the 6B, 12F and 23F type triple immune sera was significantly higher than that in the monovalent pneumococcal conjugate vaccine, and the geometric mean titer of antibodies in the remaining type triple immune sera was equivalent to that of the monovalent pneumococcal conjugate vaccine.

[0128] Table 12 Comparison of immunogenicity between monovalent vaccine and 26-valent pneumococcal conjugate vaccine

[0129] Example 11: Comparison of immunogenicity with the internationally marketed 13-valent pneumococcal conjugate vaccine (reduced amine method)

[0130] The experimental method was the same as in Example 8, with a negative control group and a test group. A similar product, the internationally marketed "13-valent pneumococcal conjugate vaccine," was used as a control substance for animal testing to compare immunogenicity. The immunization protocol employed was as follows: 1 / 4 dose of each vaccine was administered subcutaneously to NIH mice, once every two weeks for three consecutive doses. Serum collected 14 days after each dose (i.e., primary, secondary, and tertiary immunization sera) was assayed for serum-specific antibody titers. The results are shown in Table 13.

[0131] As can be seen from Table 13, the 26-valent pneumococcal conjugate vaccine is compared with the international 13-valent pneumococcal conjugate vaccine. For the 13 common types (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), the geometric mean titers of antibodies for types 4 and 19A in the triple-immune serum are slightly lower than those of the marketed 13-valent pneumococcal conjugate vaccine, but the geometric mean titers of antibodies are all within 3 times the range; the remaining 11 types are not lower than those of the marketed vaccine; for the 13 types unique to the 26-valent vaccine (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 24F, 33F, and 35B), all can stimulate mice to produce high-level antibody titers.

[0132] Table 13 Comparison of immunogenicity with 13-valent pneumococcal conjugate vaccines available internationally

[0133] Example 12 Toxicity Test - Single Dose Toxicity

[0134] Zhaoyan Pharmaceutical Research Center Co., Ltd. was commissioned to conduct a toxicity study on Sprague-Dawley rats using a single intramuscular injection of a 26-valent pneumococcal conjugate vaccine.

[0135] Experimental methods and results: Twenty SD rats (half male and half female) were used in the experiment. The animals were randomly divided into two groups, namely the negative control group and the test group. The animals in the negative control group were given 2 ml of sodium chloride injection per rat, and the animals in the test group were given 2 ml of 26-valent pneumococcal conjugate vaccine per 4 doses per rat. Multiple injections were performed on the gastrocnemius and quadriceps muscles of the bilateral hind limbs of the animals. The experimental grouping is detailed in Table 14. The animals in each group were observed continuously for at least 4 hours after administration, and then observed once in the morning and afternoon every day for a period of 14 days. During the experiment, the body weight and food intake of the animals were measured regularly, as shown in Table 15.

[0136] Table 14 Animal grouping and dose setting Note: a. The dosage unit in this study is "dose / tube". Each dose is equivalent to the clinical dose used per person per time. When the test sample is administered, if the extraction volume is less than 0.5mL, another test sample is taken and used to draw up the remaining test sample.

[0137] Table 15 Results of general toxicological index test for single-dose toxicity

[0138] The 26-valent pneumococcal conjugate vaccine was administered to SD rats by single intramuscular injection at a dose of 4 doses / rat. No animals were found to be dying / dead, and no abnormalities were found in clinical observation, body weight, or food intake. The maximum tolerated dose (MTD) was ≥ 4 doses / rat.

[0139] Example 13 Toxicity Test - Repeated Dosage Toxicity

[0140] Zhaoyan Pharmaceutical Research Center Co., Ltd. was commissioned to conduct a toxicity study on Sprague-Dawley rats using repeated intramuscular injections of a 26-valent pneumococcal conjugate vaccine for 8 weeks and a 4-week recovery period.

[0141] Experimental Methods: 120 SD rats, half male and half female, were randomly divided into four groups. During the experiment, animals in the negative control group were given 1.5 ml of sodium chloride injection; animals in the adjuvant control group were given three doses of placebo (containing aluminum phosphate adjuvant and the same content as the test article); animals in the low-dose and high-dose test article groups were given 26-valent pneumococcal conjugate vaccine, with doses of 1 and 3 doses per animal, respectively, and the administration volume was 0.5 ml per animal and 1.5 ml per animal, respectively. All animals received multiple injections into the gastrocnemius muscles of the bilateral hind limbs, once every two weeks for eight consecutive weeks, for a total of five doses (D1, D15, D29, D43, and D57). The experimental grouping details are shown in Table 16.

[0142] During the trial, the animals underwent clinical observation, body weight, food intake, body temperature, ophthalmological examination, clinical pathology (blood cell count, coagulation function, blood biochemistry, and urinalysis), immune cell phenotype (CD3+, CD3+CD4+, CD3+CD8+, and CD3+CD4+ / CD3+CD8+), and cytokine (IL-2, IL-6, TNF-α, and IFN-γ) testing. Three days after the last dose (D60), the first 10 animals per sex per group were euthanized, and the remaining animals were euthanized at the end of the 4-week recovery period (D85). All animals underwent gross autopsy, major organs were weighed, and organ-to-body ratio and organ-to-brain ratio were calculated. Histopathological examinations were performed on more than 40 tissues and organs in the negative control group, adjuvant control group, and 3-dose / animal group, as well as the injection site, skeletal muscle (biceps femoris), and inguinal lymph nodes in the 1-dose / animal group.

[0143] Table 16 Animal grouping and dose setting Note: a. The dosage unit in this study is "dose / animal". When the test article / adjuvant control group animals are dosed, the solution is extracted based on a volume of 0.5 mL per dose. If the extraction volume of one bottle is less than 0.5 mL, another bottle of test article / adjuvant control substance is used to extract the remaining test article / adjuvant control substance.

[0144] SD rats were administered a 26-valent pneumococcal conjugate vaccine by repeated intramuscular injection at doses of 1 and 3 doses per rat, administered every 2 weeks for 8 consecutive weeks (a total of 5 doses). The animals showed increases in WBC, Mono and / or Neut, Eos, FIB, and Glb, associated with immune or acute phase reactions, and decreases in HGB, MCV, MCH, Alb, and A / G. Complete recovery or a trend toward recovery was observed after the 4-week recovery period. No systemic toxicity was observed in the animals, and the no-adverse-effect level (NOAEL) was 3 doses per rat.

[0145] Under the conditions of this study, SD rats were administered 26-valent pneumococcal conjugate vaccine by repeated intramuscular injection at doses of 1 and 3 doses per rat, administered once every 2 weeks for 8 consecutive weeks, for a total of 5 doses. Local irritation reactions related to the adjuvant control substance or the test article were observed at the injection site, which recovered or showed a trend of recovery after 4 weeks of drug withdrawal. In addition, local irritation reactions related to the adjuvant control substance or the test article were observed at the injection site, which recovered or showed a trend of recovery after 4 weeks of drug withdrawal. No drug-related immunotoxic reactions were observed.

[0146] Example 14 Toxicology Test-Allergy Test

[0147] Zhaoyan Pharmaceutical Research Center Co., Ltd. was commissioned to conduct an active systemic allergic reaction test on Hartley guinea pigs with the 26-valent pneumococcal conjugate vaccine.

[0148] Experimental Methods: Thirty-six female guinea pigs were randomly divided into four groups: a negative control group, a positive control group, and low- and high-dose test article groups, with nine animals in each group. The negative control group received 0.9% sodium chloride injection; the positive control group received 20% human albumin (sensitization dose: 20 mg / animal, challenge dose: 40 mg / animal). The low- and high-dose test article groups received a 26-valent pneumococcal conjugate vaccine (sensitization and challenge doses: 0.1 and 0.2 doses / animal, respectively, in the low-dose group; 1 and 2 doses / animal, respectively, in the high-dose group). Sensitization was performed by intramuscular injection every other day for a total of three doses; challenge was performed by intravenous injection into the foot vein. Fourteen days after the final sensitization (D19), the first three animals in each group were challenged, and 21 days after the final sensitization (D26), all remaining animals in each group were challenged. After challenge, the animals in each group were observed for signs of allergic reactions. Details of the experimental groupings are shown in Table 17.

[0149] Table 17 Animal grouping and dose setting Note: The dosage unit in this study is "dose / unit", and each dose is equivalent to the clinical dose per person per time

[0150] Under the experimental conditions, the 26-valent pneumococcal conjugate vaccine was injected intramuscularly at a dose of 0.1 and 1 dose per guinea pig for sensitization, and injected intravenously at a dose of 0.2 and 2 doses per guinea pig for challenge. No active systemic allergic reaction was observed in the guinea pigs. The results are shown in Table 18.

[0151] Table 18 Statistics of allergy test results

[0152] The results of single-dose toxicity test, repeated-dose toxicity test and allergy test showed that the 26-valent pneumococcal conjugate vaccine has good safety.

[0153] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A 26-valent pneumococcal conjugate combination vaccine, characterized in that, Pneumococcal capsular polysaccharides include: 26 serotypes, namely 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B, and the carrier protein is TT.

2. The vaccine according to claim 1, wherein Pneumococcal capsular polysaccharides other than serotype 2 and serotype 3 are activated with 1-cyano-4-dimethylaminopyridine tetrafluoroborate, and then form derivatives with adipic dihydrazide (ADH) as a spacer, and then react with the carrier protein TT under the action of carbodiimide (EDAC) to generate polysaccharide-protein conjugates.

3. The vaccine according to claim 1, wherein Serotype 2 and serotype 3 polysaccharides are activated with CDAP and directly combined with TT to generate polysaccharide-protein conjugates.

4. The vaccine according to claim 1, characterized in that, The vaccine also contains an adjuvant, and the adjuvant is an aluminum adjuvant, and the aluminum adjuvant is an aluminum phosphate adjuvant.

5. The vaccine according to claim 1, characterized in that, The weight-average molecular weight Mw of most types of pneumococcal polysaccharides is 300 - 800 kDa, Mw of serotype 2 and serotype 7F is 900 - 1200 kDa, and Mw of serotype 18C, 19A and 19F is 100 - 400 kDa.

6. The vaccine according to claim 1, characterized in that, The derivation rate of pneumococcal polysaccharide derivatives of serotype 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B is 1% - 10%.

7. The vaccine according to claim 1, characterized in that, The free polysaccharide in the stock solution of the polysaccharide-protein conjugate of serotype 4 is not higher than 45%, the free polysaccharide in the stock solutions of the polysaccharide-protein conjugates of the other 25 serotypes is not higher than 30%, and the free protein is not higher than 5%.

8. The vaccine according to claim 1, characterized in that, In the combined vaccine of unit dose, the content of each type of pneumococcal polysaccharide is 2.2 μg ± 30%, and that of 6B is 4.4 μg ± 30%; The pH of the vaccine is 5.0 - 7.0, the aluminum ion content is 0.15 - 0.35 mg / ml, the sodium ion content is 7.5 - 9.5 g / L, and the content of Tween 80 is 120 - 180 μg / ml.

9. The method for preparing the vaccine according to claim 1, characterized in that, It includes the following steps: Step 1: After the pneumococcal polysaccharides are fermented by strains and the bacteria are removed, the polysaccharide purification process uses CTAB precipitation, sodium chloride dissociation, hydroxyapatite chromatography, and finally freeze-drying to obtain refined pneumococcal polysaccharides; Step 2: Pneumococcal polysaccharides of serotype 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B are activated with CDAP, undergo a derivation reaction with ADH as a spacer, form polysaccharide derivatives after ultrafiltration purification, and then undergo a condensation reaction with the carrier protein TT under the action of EDAC, and obtain polysaccharide-protein conjugates through ultrafiltration; Step 3: Serotype 2 and serotype 3 polysaccharides are activated with CDAP and directly react with TT, and polysaccharide-protein conjugates are obtained through ultrafiltration; Step 4: The polysaccharide-protein conjugate is chromatographed on Sepharose 4FF, and the fractions near V0 and before K D 0.2 are the purified conjugate. After sterile filtration, it is the stock solution of the pneumococcal polysaccharide-protein conjugate; Step 5: Mix the stock solution of the 26-valent polysaccharide-protein conjugate, add Tween 80, aluminum phosphate adjuvant, and sodium chloride solution, and fill it to obtain the 26-valent pneumococcal conjugate vaccine.

10. The method for preparing the vaccine according to claim 9, wherein in Step 2, wherein, the mass ratio of CDAP to polysaccharide is 0.1 - 1.0, preferably 0.1 - 0.5 for serotypes 8, 9N, 9V, 10A, 20, 33F, and 35B, and 0.5 - 1.0 for other serotypes; wherein, the final concentration of ADH is 0.2 mol / L; wherein, the pH during the reaction process is 8.0 - 9.0; wherein, the reaction time is not less than 2 hours; in Step 3, wherein, the mass ratio of CDAP to polysaccharide is 0.1 - 1.0, preferably 0.25 - 0.75; wherein, the mass ratio of polysaccharide to carrier protein TT is 1:0.5 - 1:2, preferably 1:1 - 1:2; wherein, the pH during the reaction process is 8.0 - 9.0, wherein, the reaction time is not less than 2 hours.

11. Use of the vaccine according to any one of claims 1 - 8 in the preparation of a medicament for preventing or treating pneumonia.

12. The vaccine according to any one of claims 1 - 8 for use in preventing or treating.

13. The vaccine according to any one of claims 1 - 8 for use in preventing or treating pneumonia.

14. A method for preventing or treating pneumonia, which comprises administering to a subject a prophylactically or therapeutically effective amount of the vaccine according to any one of claims 1 - 8.

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