31-valent pneumococcus capsular polysaccharide composition and application thereof

Abstract

The invention provides a composition of 31-valent pneumococcal capsular polysaccharide and application thereof, the composition comprises 31 pneumococcal serotype capsular polysaccharide, and the capsular polysaccharide and carrier protein form a conjugate. By integrating a multi-carrier compatibility strategy, an adjuvant-free preparation technology and serotype specific antigen dosage optimization, the safety and immune protection effect of the vaccine are remarkably improved. The vaccine effectively overcomes carrier inhibition in multivalent vaccines, adopts an adjuvant-free formula to thoroughly avoid local and systemic adverse reactions related to aluminum adjuvants, improves inoculation compliance, performs antigen dosage enhancement on low immunogenicity serotypes, and synergistically enhances overall immune response breadth and strength. The product is stable in process, suitable for large-scale production and capable of providing safer and broad-spectrum protection for sensitive people such as infants and old people.

Description

[0001] This application claims priority to Chinese patent application 2025106757070, filed on May 23, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to compositions of 31-valent pneumococcal capsular polysaccharides and their applications. Background Technology

[0003] Streptococcus pneumoniae Streptococcus pneumoniae is a common bacterium belonging to the genus Streptococcus. It is one of the main pathogens causing pneumonia and can also cause other infections such as otitis media, sinusitis, and meningitis. Based on capsular polysaccharide serotypes, there are over 100 serotypes of Streptococcus pneumoniae, some of which are highly pathogenic. This bacterium is usually found in the nasopharynx of humans and is transmitted through droplets. When a person's immunity is weakened, Streptococcus pneumoniae may invade the lower respiratory tract, causing pneumonia. Symptoms of pneumococcal pneumonia include high fever, chills, cough, sputum production, and chest pain; in severe cases, it can lead to respiratory failure. Children, the elderly, immunocompromised individuals, and people with chronic diseases are more susceptible to Streptococcus pneumoniae infection.

[0004] Currently, various pneumococcal conjugate vaccines are available on the market, such as 7-valent, 13-valent, 20-valent, and 21-valent vaccines. These vaccines are widely used globally and have significantly reduced the incidence of pneumococcal disease. For example, the 7-valent pneumococcal conjugate vaccine was widely administered and effectively prevented infections caused by specific serotypes of Streptococcus pneumoniae. The 13-valent pneumococcal conjugate vaccine further expanded its protection, covering more common pathogenic serotypes and being included in immunization programs, playing an important role in public health. Marketed pneumococcal conjugate vaccines have shown significant effectiveness in preventing Streptococcus pneumoniae infection. However, with the widespread use of these vaccines, serosubstitution has gradually emerged. Serosubstitution refers to the phenomenon where, after vaccination, while infection with the vaccine-targeted serotype of Streptococcus pneumoniae is effectively controlled, infections caused by non-vaccine serotypes of Streptococcus pneumoniae relatively increase, which to some extent weakens the overall protective efficacy of existing vaccines. Furthermore, the prevalence of strains not covered by existing vaccines is increasing.

[0005] Market demand for pneumococcal conjugate vaccines is trending towards higher valences. Theoretically, higher valence vaccines can cover more pneumococcal serotypes, thus providing broader immune protection. However, increasing valence is not without risk. On the one hand, as valence increases, the complexity of the vaccine manufacturing process increases significantly, potentially leading to decreased product quality stability. On the other hand, the combination of multiple antigenic components may trigger immune competition, affecting vaccine efficacy. For example, different serotype antigens may interfere with each other during the immune response, resulting in insufficient antibody levels against certain serotypes and failing to achieve the desired protective effect.

[0006] From a safety perspective, increasing the antigen content may lead to a higher risk of adverse reactions. From an efficacy perspective, appropriately increasing the antigen content can improve antibody levels, but beyond a certain point, further increases in antigen content will have limited immunizing effects. Furthermore, the increased antigen content may lead to an increase in carrier protein content, causing carrier inhibition and thus reducing immunogenicity. From a process perspective, high antigen content requires high-concentration stock solutions when preparing semi-finished products, and high-concentration stock solutions have higher viscosity and poorer stability. From a cost perspective, increasing the antigen content means higher costs.

[0007] In summary, there is an urgent need in this field for a new generation of high-valent pneumococcal conjugate vaccines that can simultaneously address: the vector inhibition effect of multivalent pneumococcal conjugate vaccines; some safety issues caused by adjuvants; and the challenge of insufficient immunogenicity of weak serotypes. Summary of the Invention

[0008] To address the aforementioned technical challenges, this invention proposes a multi-carrier compatibility strategy, an adjuvant-free dosage form strategy, and a serotype-specific antigen dosage adjustment strategy, providing a multivalent pneumococcal conjugate vaccine. This approach enhances the overall immune response and protective spectrum of the vaccine by matching the optimal carrier protein to different serotypes, optimizing the formulation process in an adjuvant-free system to ensure antigen stability, and specifically increasing the antigen content of low-immunogenic serotypes. This achieves a broader and more balanced protective effect without sacrificing safety.

[0009] In one aspect, this invention provides a composition of 31-valent pneumococcal capsular polysaccharides, the composition comprising 31 species of pneumococci. The serological capsular polysaccharides are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B. Among them, the content of capsular polysaccharides of individual types 1, 3, 6B, 19A, and 19F in the composition is higher than that of other types.

[0010] In some implementations, the capsular polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B are hydrolyzed capsular polysaccharides.

[0011] In some embodiments, the composition is divided into three groups based on the content of individual capsular polysaccharides:

[0012] Group 1): Capsular polysaccharides of types 1, 3, 6B, 19A, and 19F;

[0013] 2) Groups: Capsular polysaccharides of types 15A, 15C, 16F, 23A, 23B, 31, and 35B; and,

[0014] 3) Groups: Capsular polysaccharides of types 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F and 33F; among them, the content of capsular polysaccharides of different serotypes within the same group is the same, while the content of capsular polysaccharides of different types varies between groups.

[0015] The term “polysaccharide” is intended to include any antigenic sugar element (or antigenic unit) commonly used in the fields of immunization and bacterial vaccines, including but not limited to “sugars,” “oligosaccharides,” “polysaccharides,” “liposaccharides,” “lipo-oligosaccharides (LOS),” “lipopolysaccharides (LPS),” “glycosylations,” “glycoconjugates,” etc.

[0016] In some embodiments, for a single type of capsular polysaccharide in the composition, the capsular polysaccharide content in group 1) is 2-6 times that in group 2); and / or, the capsular polysaccharide content in group 3) is 1-3 times that in group 2).

[0017] In the composition, for a single type of capsular polysaccharide, the capsular polysaccharide content in group 1) is 4-5 times that in group 2); and / or, the capsular polysaccharide content in group 3) is 2 times that in group 2).

[0018] In some embodiments, the composition satisfies one or more of the following conditions:

[0019] a. 1) The content of a single capsular polysaccharide in the group is 4~10 μg / 0.5 mL; for example, 10 μg / 0.5 mL, 8 μg / 0.5 mL, 6 μg / 0.5 mL or 4 μg / 0.5 mL;

[0020] b. 2) The content of single-type capsular polysaccharides in group 1.5~2.5μg / 0.5mL; and,

[0021] c. 3) The content of capsular polysaccharide of a single type in group is 3.5~4.5μg / 0.5mL.

[0022] In some embodiments, the composition satisfies one or more of the following conditions:

[0023] a. The capsular polysaccharide content of serotypes 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F and 33F in the composition is 4 μg / 0.5 mL;

[0024] b. The capsular polysaccharide content of serotypes 15A, 15C, 16F, 23A, 23B, 31 and 35B in the composition is 2 μg / 0.5 mL;

[0025] c. The content of capsular polysaccharides of serotypes 1, 3, 6B, 19A and 19F in the composition is 4 to 10 μg / 0.5 mL; for example, 10 μg / 0.5 mL, 8 μg / 0.5 mL, 6 μg / 0.5 mL or 4 μg / 0.5 mL.

[0026] In some embodiments, the content of a single capsular polysaccharide in group 1) of the composition is 8-10 μg / 0.5 mL, the content of a single capsular polysaccharide in group 2) is 2 μg / 0.5 mL, and the content of a single capsular polysaccharide in group 3) is 4 μg / 0.5 mL.

[0027] In some embodiments, the composition further comprises a carrier protein, wherein the capsular polysaccharide forms a conjugate with the carrier protein.

[0028] In some embodiments, the composition does not contain an adjuvant.

[0029] In some embodiments, the carrier protein is selected from one or more of pneumococcal hemolysin (rPly), tetanus toxoid (TT), diphtheria toxin non-toxic mutant (rCRM197), and recombinant pneumococcal histidine triplet protein (rPhtD).

[0030] In some embodiments, the tetanus toxoid is the tetanus toxoid C fragment (rTTc).

[0031] In some embodiments, the carrier protein includes pneumococcal hemolysin and / or tetanus toxin C fragment; it also includes one or more of tetanus toxoid, a non-toxic mutant of diphtheria toxin, and recombinant pneumococcal histidine triplet protein.

[0032] In some implementations, the carrier protein includes pneumococcal hemolysin, tetanus toxin C fragment, and a non-toxic mutant of diphtheria toxin.

[0033] In some embodiments, the pneumococcal hemolysin is recombinant pneumococcal hemolysin.

[0034] In some embodiments, the tetanus toxin C fragment is a recombinant tetanus toxin C fragment.

[0035] In some embodiments, the diphtheria toxin non-toxic mutant is the recombinant diphtheria toxin non-toxic mutant CRM197.

[0036] In some embodiments, the capsular polysaccharides of serotypes 1, 3, 6B, 19A, and 19F in the composition are conjugated with pneumococcal hemolysin.

[0037] In some embodiments, one or more capsular polysaccharides from the 31 serotypes in the composition are conjugated with pneumococcal hemolysin.

[0038] In some embodiments, one or more capsular polysaccharides from the 31 serotypes in the composition are conjugated with a non-toxic mutant of diphtheria toxin.

[0039] In some embodiments, one or more capsular polysaccharides from 31 serotypes in the composition are conjugated to a tetanus toxin C fragment.

[0040] In some embodiments, the 31 serotypes of capsular polysaccharides in the composition are divided into two or three groups, with different carrier proteins used for conjugation between the groups.

[0041] In some specific embodiments, the 31 serotypes of capsular polysaccharides in the composition are divided into the following three groups:

[0042] Group i) Contains one or more of the serotypes 1, 3, 4, 6A, 6B, 11A, 14, 17F, 19A, 19F, 20, 22F, 23B, 23F, 31 and 35B of capsular polysaccharides; preferably containing 9 to 13 types, such as 10, 11 or 13 types;

[0043] ii) Group: comprising one or more of serotypes 1, 2, 5, 7F, 8, 9V, 9N, 10A, 11A, 14, 15A, 15B, 15C, 16F, 18C, 20, 31, and 33F capsular polysaccharides; preferably comprising 10-13, for example 11 or 12; and,

[0044] iii) Group: Contains one or more of the capsular polysaccharides of serotypes 3, 5, 7F, 8, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 22F, 23A and 23B; preferably contains 5-11 types, such as 6, 9 or 10 types; wherein, the carrier protein bound to the single-type capsular polysaccharide is the same for different serotypes within the group, and the carrier protein bound to the single-type capsular polysaccharide is different between groups; and the single-type capsular polysaccharide appears only once in the three groups.

[0045] In some embodiments, the composition satisfies one or more of the following conditions:

[0046] i) The capsular polysaccharides in group i) were conjugated with pneumococcal hemolysin, respectively;

[0047] ii) The capsular polysaccharides in group ii) were conjugated with the diphtheria toxin nontoxic mutant, respectively; and,

[0048] In group iii), the capsular polysaccharides were conjugated to the tetanus toxin C fragment.

[0049] In some embodiments, the composition is selected from any of the following:

[0050] 1) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23B, 23F, and 35B with pneumococcal hemolysin; 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 18C, 31, and 33F with non-toxic mutants of diphtheria toxin; 5) Conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 12F, 22F, and 23A with the C fragment of tetanus toxin.

[0051] 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 20, 23B, 23F, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 18C, 19A, 20, 23A, and 31 with diphtheria toxin non-toxic mutants; and conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, and 14 with tetanus toxin C fragment.

[0052] 3) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23A, 23B, and 23F with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 11A, 15A, 15B, 15C, 16F, 18C, 31, 33F, and 35B with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, and 22F with the C fragment of tetanus toxin.

[0053] 4) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 23B, 23F, 31, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 11A, 15B, 15C, 16F, and 18C with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment;

[0054] 5) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6A, 6B, 14, 17F, 19F, 23B, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 9V, 9N, 11A, 15B, 15C, 16F, 18C, and 33F with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment;

[0055] 6) Conjugates formed by conjugating capsular polysaccharides of serotypes 4, 6A, 6B, 11A, 17F, 19F, 22F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 5, 7F, 8, 14, 15A, 15C, 16F, 20, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 3, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 23A, and 23B with tetanus toxin C fragment; and,

[0056] 7) Conjugates formed by conjugating capsular polysaccharides of serotypes 3, 4, 6A, 6B, 11A, 14, 17F, 19F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 9V, 10A, 15A, 15B, 15C, 16F, 18C, 20, and 33F with non-toxic mutants of diphtheria toxin; and conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 9N, 12F, 19A, 22F, 23A, and 23B with tetanus toxin C fragment.

[0057] Purification methods for pneumococcal capsular polysaccharides are also conventional techniques in this field, such as those achieved through solvent-free methods. Typical polysaccharide purification processes include, but are not limited to, dissolving the complex sugar in NaCl solution, centrifuging after dissolution in NaCl solution, collecting the supernatant, adding anhydrous ethanol to remove impurities and precipitated sugar, washing the precipitate multiple times to remove impurities, or, depending on the different sugar forms, using chromatography methods such as ion exchange columns or composite packing materials to obtain purified polysaccharides. In a specific implementation, the purification process mainly includes separating the bacterial cells by centrifugation of the bacterial inactivation solution, collecting the supernatant, purifying it using an ion exchange chromatography column, concentrating it through a 10 kDa membrane, replacing it, desalting it, and then lyophilizing it to obtain the refined polysaccharide.

[0058] The polysaccharide-protein conjugates of the present invention can be formulated using methods well-known in the art. For example, single pneumococcal conjugates can be formulated to prepare compositions using physiologically acceptable carriers. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

[0059] The polysaccharide-protein conjugates of this invention can be generated using various conjugation methods. The polysaccharide-protein binding reaction typically includes polysaccharide activation and protein conjugation steps. Chemical activation of the polysaccharide followed by conjugation to the carrier protein is achieved using conventional methods. By controlling parameters such as the molecular weight of the polysaccharide, the polysaccharide activation reaction, the polysaccharide-protein reaction ratio, the reaction temperature, the reaction time, and the pH of the reaction solution, the final polysaccharide-protein conjugate is obtained. The obtained polysaccharide-protein conjugate is further purified to remove unreacted substances and impurities. Quality analysis is used to quantify its polysaccharide-protein cross-linking degree and residual impurities, ensuring batch consistency and comparability.

[0060] The purified polysaccharide is chemically activated to enable it to react with carrier proteins. Different chemical activation methods can be selected based on the characteristics of the polysaccharide. Commonly used methods include cyanogen bromide oxidation (US6375846B1), hydrolysis (US4761283), 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (EP0720485), and periodic acid oxidation (US4711779). Activated polysaccharides can be separated from other impurities by ultrafiltration. After activation, the polysaccharide exhibits reactivity with carrier proteins.

[0061] In some embodiments, the pneumococcal capsular polysaccharide and recombinant pneumococcal hemolysin are promoted to form a conjugate via CDAP. This can be achieved through the following steps: a. Dissolving the hydrolyzed pneumococcal capsular polysaccharide in a salt solution and adjusting the pH to 8.0–10.0; b. Adding CDAP solution and adjusting the pH to 8.0–10.0; for example, using triethylamine to adjust the pH; c. Adding a carrier protein and reacting for 2–24 hours; d. After the reaction, diluting with 0.85% NaCl solution, purifying using an ultrafiltration membrane, and collecting the retentate to obtain the conjugate.

[0062] CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is a chemical reagent that acts as a cross-linking agent to promote the binding of polysaccharides to proteins. In the preparation of vaccines and other biopharmaceuticals, cross-linking agents are used to link different biomolecules together to enhance vaccine stability and immunogenicity.

[0063] After capsular polysaccharides are conjugated to carrier proteins, the polysaccharide-protein conjugates are purified using a variety of techniques. These techniques include concentration / percolation, precipitation / elution, and column chromatography.

[0064] After purifying the various polysaccharide-protein conjugates, they are mixed to formulate the compositions of the present invention, which can be used as vaccines. The formulations of the compositions of the present invention can be accomplished using methods known in the art. For example, 31 individual pneumococcal conjugates can be formulated to prepare the compositions using pharmaceutically acceptable excipients. Examples of such excipients include, but are not limited to, water, buffered saline, amino acids (e.g., arginine, proline, etc.), surfactants (e.g., polysorbates, poloxamer, etc.), and excipients (e.g., trehalose, sucrose, etc.).

[0065] In some embodiments, the composition does not contain capsular polysaccharide-protein conjugates derived from any other pneumococcal serotype.

[0066] In some embodiments, the pneumococcal hemolysin has an amino acid sequence as shown in SEQ ID NO: 1.

[0067] In some embodiments, the tetanus toxin C fragment has an amino acid sequence as shown in SEQ ID NO: 2.

[0068] In some embodiments, the diphtheria toxin nontoxic mutant has an amino acid sequence as shown in SEQ ID NO: 3.

[0069] In some embodiments, the ratio of capsular polysaccharide to carrier protein in the composition is (0.5-1.5):1, preferably 1:1.

[0070] In some implementations, the capsular polysaccharide is hydrolyzed (except for type 19A, which is not hydrolyzed) and then activated with the carrier protein via CDAP to form a conjugate.

[0071] In some embodiments, the tetanus toxin C fragment is prepared by alkylation modification; or by cysteine ​​mutation; or by a natural protein.

[0072] The dosage of the composition should be a dose that achieves an immunogenic effect. The amount of conjugate in each dose of the composition is selected as the amount that induces immune protection without significant adverse effects. This amount can vary depending on the pneumococcal serotype. Typically, each dose will contain 0.01 μg to 100 μg of polysaccharide or conjugate, for example, 0.1 μg to 10 μg, or 4 μg to 10 μg. The administered dosage is generally in the range of about 0.01 μg to about 10 μg per kilogram of body weight. A series of optimal immunizing doses can be given. A unit dosage form of the composition may contain an equivalent amount of meningococcal capsular polysaccharide or conjugate, for example, 0.1 μg to 10 μg, or 4 μg to 10 μg.

[0073] Another aspect of the present invention provides a vaccine comprising an effective amount of the composition as described in one aspect of the present invention; the vaccine is used to induce an immune response against pneumococcus, and the vaccine further comprises excipients.

[0074] The compositions and vaccines of the present invention can typically be formed by dispersing pneumococcal capsular polysaccharides or conjugates into suitable pharmaceutically acceptable excipients. These excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, and preservatives. The excipients are preferably non-toxic to the recipient at the doses and concentrations used, and are, for example, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0075] In some embodiments, the excipients comprise one or more of sodium chloride, sodium acetate, citric acid, polysorbate 80, trehalose, sucrose, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and arginine.

[0076] In some embodiments, the excipient comprises one or more of sodium chloride, sodium acetate, and polysorbate 80.

[0077] In some embodiments, the sodium chloride content in the vaccine is 3.75~4.75 mg / 0.5 mL.

[0078] In some implementations, the vaccine contains 4.5 mg of sodium chloride per 0.5 mL.

[0079] In some implementations, the sodium acetate content in the vaccine is 1-3 mg / 0.5 mL.

[0080] In some embodiments, the vaccine contains 1.23 mg of sodium acetate per 0.5 mL.

[0081] In some embodiments, the content of polysorbate 80 in the vaccine is 80~2500 μg / 0.5 mL.

[0082] In some embodiments, the vaccine contains 100 μg / 0.5 mL of polysorbate 80.

[0083] In some implementations, the vaccine does not contain an adjuvant.

[0084] In some embodiments, the capsular polysaccharide content of the vaccine for serotypes 15A, 15C, 16F, 23A, 23B, 31, and 35B is 2 μg / 0.5 mL; the capsular polysaccharide content for serotypes 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F is 4 μg / 0.5 mL; and the capsular polysaccharide content for serotypes 1, 3, 6B, 19A, and 19F is 4–10 μg / 0.5 mL.

[0085] In some embodiments, the content of monotype capsular polysaccharides of types 15A, 15C, 16F, 23A, 23B, 31, and 35B in the vaccine is 2 μg / 0.5 mL; the content of monotype capsular polysaccharides of types 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F is 4 μg / 0.5 mL; and the content of monotype capsular polysaccharides of types 1, 3, 6B, 19A, and 19F is 8-10 μg / 0.5 mL.

[0086] In some embodiments, the capsular polysaccharide content of the vaccine for serotypes 15A, 15C, 16F, 23A, 23B, 31, and 35B is 2 μg / 0.5 mL; for serotypes 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F, it is 4 μg / 0.5 mL; and for serotypes 1, 3, 6B, 19A, and 19F, it is 10 μg / 0.5 mL.

[0087] In some embodiments, the vaccine comprises conjugates formed by combining capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23F, 23B, and 35B with pneumococcal hemolysin; conjugates formed by combining capsular polysaccharides of serotypes 2, 9V, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 18C, 31, and 33F with a non-toxic mutant of diphtheria toxin; and conjugates formed by combining capsular polysaccharides of serotypes 5, 7F, 8, 12F, 22F, and 23A with a tetanus toxin C fragment. The vaccine contains 2 μg / 0.5 mL of capsular polysaccharide for serotypes 15A, 15C, 16F, 23A, 23B, 31, and 35B; 4 μg / 0.5 mL of capsular polysaccharide for serotypes 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F; and 10 μg / 0.5 mL of capsular polysaccharide for serotypes 1, 3, 6B, 19A, and 19F. The excipients in the vaccine are sodium acetate, polysorbate 80, and sodium chloride.

[0088] In some implementations, the vaccine contains:

[0089] 1) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23B, 23F, and 35B with pneumococcal hemolysin; 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 18C, 31, and 33F with non-toxic mutants of diphtheria toxin; 3) Conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 12F, 22F, and 23A with the C fragment of tetanus toxin.

[0090] 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 20, 23B, 23F, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 18C, 19A, 20, 23A, and 31 with diphtheria toxin non-toxic mutants; and conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, and 14 with tetanus toxin C fragment.

[0091] 3) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23A, 23B, and 23F with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 11A, 15A, 15B, 15C, 16F, 18C, 31, 33F, and 35B with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, and 22F with the C fragment of tetanus toxin.

[0092] 4) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 23B, 23F, 31, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 11A, 15B, 15C, 16F, and 18C with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment;

[0093] 5) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6A, 6B, 14, 17F, 19F, 23B, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 9V, 9N, 11A, 15B, 15C, 16F, 18C, and 33F with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment;

[0094] 6) Conjugates formed by conjugating capsular polysaccharides of 4, 6A, 6B, 11A, 17F, 19F, 22F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of 1, 2, 5, 7F, 8, 14, 15A, 15C, 16F, 20, and 33F with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of 3, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 23A, and 23B with tetanus toxin C fragment;

[0095] 7) Conjugates formed by conjugating capsular polysaccharides of serotypes 3, 4, 6A, 6B, 11A, 14, 17F, 19F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 9V, 10A, 15A, 15B, 15C, 16F, 18C, 20, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 9N, 12F, 19A, 22F, 23A, and 23B with tetanus toxin C fragment;

[0096] 8) Conjugates formed by combining capsular polysaccharides of 31 serotypes with pneumococcal hemolysin;

[0097] 9) Conjugates formed by combining capsular polysaccharides from 31 serotypes with non-toxic mutants of diphtheria toxin; or,

[0098] 10) Conjugates formed by combining capsular polysaccharides of 31 serotypes with tetanus toxin C fragments.

[0099] In some implementations, the vaccine meets one or more of the following criteria:

[0100] 1) The capsular polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B are hydrolyzed capsular polysaccharides;

[0101] 2) The pneumococcal hemolysin has the amino acid sequence shown in SEQ ID NO: 1;

[0102] 3) The tetanus toxin C fragment has the amino acid sequence shown in SEQ ID NO: 2;

[0103] 4) The diphtheria toxin non-toxic mutant has the amino acid sequence shown in SEQ ID NO: 3; and,

[0104] 5) The ratio of capsular polysaccharide to carrier protein in the composition is (0.5-1.5):1, preferably 1:1.

[0105] In some implementations, the vaccine is in liquid form.

[0106] The vaccine of the present invention is a pharmaceutical preparation containing more than one active agent (e.g., pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) that provides active immunity against diseases or pathological conditions caused by more than one serotype of Streptococcus pneumoniae.

[0107] Another aspect of the present invention provides the use of the composition provided in one aspect of the present invention in the preparation of a medicament for the prevention of pneumococcal-related diseases; preferably, the medicament is a vaccine.

[0108] Another aspect of the present invention provides a method for preparing the composition as described herein, the method comprising the following steps:

[0109] 1) Conjugating serum-type capsular polysaccharide to a carrier protein to obtain a conjugate; and,

[0110] 2) Mix the conjugates obtained in step 1) to obtain a composition.

[0111] In some embodiments, in step 1), the capsular polysaccharide is activated and then conjugated to the carrier protein. The activation is chemical activation, such as cyanogen bromide method, CDAP method or periodic acid oxidation method. Preferably, a hydrolysis step is also included before the activation of the capsular polysaccharide, such as acid hydrolysis, hot water hydrolysis or enzymatic hydrolysis.

[0112] The preparation of conjugates involves chemically coupling polysaccharide fragments obtained by processing purified and extracted pneumococcal outer membrane polysaccharides to a carrier protein. Polysaccharide-protein conjugates can be obtained through direct chemical coupling or via linkers, such as adipic acid dihydrazide. Various chemical methods for conjugating polysaccharides to proteins are known and described in the literature. Common methods include the cyanogen bromide method, the CDAP method, or the reductive amination method (US5952454, EP0720485, US4711779). Polysaccharide-protein conjugates obtained through coupling can induce relatively strong immunogenicity, simultaneously inducing specific antibodies against both the polysaccharide and the protein.

[0113] Another aspect of the present invention provides a method for preparing a pneumococcal vaccine, the method comprising the step of adding excipients to the composition provided by the present invention.

[0114] In some implementations, the method further includes a sterilization step, such as sterilization by filtration.

[0115] In some embodiments, the excipients are as defined in the vaccines provided by this invention.

[0116] Another aspect of the present invention provides a method for generating specific antibodies in vivo, the method comprising administering an effective amount of the composition provided by the present invention or the vaccine provided by the present invention to a subject in need.

[0117] Another aspect of the present invention provides a method for inducing an immune response in a human patient, comprising administering to the patient an effective amount of the composition or vaccine provided by the present invention.

[0118] In some implementations, the immune response is a protective immune response.

[0119] In some implementations, the protective immune response is a protective immune response against Streptococcus pneumoniae.

[0120] Another aspect of the present invention provides a method for preventing diseases, symptoms or illnesses caused by Streptococcus pneumoniae, comprising administering an effective amount of the composition or vaccine provided by the present invention to a subject in need.

[0121] In some implementations, the disease, condition, or symptom is selected from at least one of the following:

[0122] Common pneumococcal disease, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, infiltrative diseases caused by Streptococcus pneumoniae, and otitis media caused by Streptococcus pneumoniae.

[0123] The compositions or vaccines provided by this invention are used to prevent diseases, symptoms, or illnesses caused by Streptococcus pneumoniae.

[0124] In some implementations, the disease, condition, or symptom is selected from at least one of the following:

[0125] Common pneumococcal disease, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, infiltrative diseases caused by Streptococcus pneumoniae, and otitis media caused by Streptococcus pneumoniae.

[0126] The composition or vaccine provided by this invention may be used in the following ways:

[0127] 1) Disease prevention;

[0128] 2) Inhibits Streptococcus pneumoniae infection;

[0129] 3) Inducing an immune response or protective immune response against Streptococcus pneumoniae;

[0130] 4) Prevention of Streptococcus pneumoniae infection;

[0131] 5) Prevent recurrence of pneumococcal infection;

[0132] 6) Reduce the progression, onset, or severity of pathological symptoms associated with Streptococcus pneumoniae infection, including prevention of related complications such as brain damage, hearing loss, and seizures; and,

[0133] 7) Reduces the likelihood of pneumococcal infection.

[0134] The use of the composition or vaccine provided by this invention in the preparation of a medicament having the following functions:

[0135] 1) Disease prevention;

[0136] 2) Inhibits Streptococcus pneumoniae infection;

[0137] 3) Inducing an immune response or protective immune response against Streptococcus pneumoniae;

[0138] 4) Prevention of Streptococcus pneumoniae infection;

[0139] 5) Prevent recurrence of pneumococcal infection;

[0140] 6) Reduce the progression, onset, or severity of pathological symptoms associated with Streptococcus pneumoniae infection, including prevention of related complications such as brain damage, hearing loss, and seizures; and,

[0141] 7) Reduces the likelihood of pneumococcal infection.

[0142] The “effective amount” of the composition of the present invention refers to the dose of stimulating antibody that significantly reduces the likelihood or severity of microbial (e.g., Streptococcus pneumoniae) infection during subsequent challenge.

[0143] "Patient" (which may be referred to herein as "subject") means a mammal capable of being infected with Streptococcus pneumoniae. In a preferred embodiment, the patient is a human. Patients may be given preventative or therapeutic treatment. Prophylactic treatment provides sufficient protective immunity to reduce the likelihood or severity of pneumococcal infection or its effects (e.g., pneumococcal pneumonia). Therapeutic treatment may be given to reduce the severity of Streptococcus pneumoniae infection or its clinical effects or to prevent its recurrence. As described herein, prophylactic treatment can be performed using the multivalent immunogenic compositions of the present invention. The compositions of the present invention can be administered to the general population or to those at increased risk of pneumococcal infection, such as the elderly, or to persons living with or caring for the elderly.

[0144] "Subjects in need" include people who have been previously exposed to or infected with Streptococcus pneumoniae, people who have been previously vaccinated against Streptococcus pneumoniae, and people who are susceptible to infection or need to reduce their chance of infection, such as people with weakened immune systems, the elderly, children, adults, or healthy individuals.

[0145] Administration may include one or more of the following: intramuscular, intraperitoneal, intradermal, or subcutaneous injection; or administration via mucosal route to the oral / gastrointestinal tract, respiratory tract, or genitourinary tract. In one embodiment, intranasal administration may be used to treat pneumonia or otitis media (because it can more effectively prevent nasopharyngeal transport of pneumococcus, thus reducing infection in its early stages). In certain embodiments, the compositions of the invention are administered to the patient via intramuscular or subcutaneous administration.

[0146] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0147] The reagents and raw materials used in this invention are all commercially available.

[0148] The positive and progressive effects of this invention are as follows:

[0149] Vaccines containing the 31-valent pneumococcal capsular polysaccharide of the present invention can effectively reduce vector epitope inhibition, thereby significantly enhancing immunogenicity.

[0150] Furthermore, the adjuvant-free formulation, by eliminating aluminum phosphate adjuvant, greatly enhances vaccine safety.

[0151] Furthermore, increasing the dosage of antigens for serotypes 1, 3, 6B, 19A, and 19F not only significantly enhanced the immunogenicity of these serotypes themselves, but also exhibited a significant synergistic immune effect when used in combination with recombinant pneumococcal hemolysin (Ply), further improving antibody responses to other serotypes in the vaccine and thus broadening the overall immunoprotective spectrum of the vaccine. This, in turn, opens up possibilities for the development and application of higher-valence vaccine compositions, enabling them to be effectively utilized in the preparation of vaccines with more valences. Detailed Implementation

[0152] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0153] Example 1: Preparation of hydrolyzed capsular polysaccharide / capsular polysaccharide

[0154] Capsular polysaccharides from *Streptococcus pneumoniae* can be prepared using standard techniques known to those skilled in the art. *Streptococcus pneumoniae* bacterial culture / fermentation and polysaccharide preparation is a well-established process (US4686102, US5847112). Different serotypes of *Streptococcus pneumoniae* (from the American Type Culture Collection) are cultured to prepare a strain library. One tube of *Streptococcus pneumoniae* strain from the library is inoculated into a shake flask containing soybean medium and incubated overnight at 37°C and 5–10% CO2. After incubation, microscopic examination is performed. If the results are normal, the culture is inoculated into a fermenter and fermented at pH 7.0 and 37°C. After incubation, 10% sodium deoxycholate is added for sterilization to ensure complete inactivation of the *Streptococcus pneumoniae* bacteria. The pH was adjusted to 3.0-7.0 with acid and stirred for 1 hour. The acid-precipitated sample was clarified using a 0.2 μm microfiltration membrane. The clarified permeate was concentrated and washed with a 10-100 kDa membrane. The sample after replacement was subjected to ion exchange column chromatography. The flow-through was collected and concentrated and washed with a 10-100 kDa membrane to obtain purified polysaccharides.

[0155] Different types of pneumococcal capsular polysaccharides were prepared for serotype identification using specific sera, chemical structure analysis using nuclear magnetic resonance (NMR) spectroscopy, and determination of functional groups (such as rhamnose, uronic acid, and O-acetyl groups) using chemical colorimetric methods. The polysaccharide quality was confirmed to meet WHO polysaccharide quality standards.

[0156] Capsular polysaccharides of types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B, obtained after fermentation and purification, can be hydrolyzed using the method described in patent US5847112A to obtain hydrolyzed capsular polysaccharides. Alternatively, the purified polysaccharides can be dissolved in pure water to a concentration of 5 mg / mL. The magnetic stirring speed is adjusted to 400 rpm, and the magnetic stirrer size is A60. At the beginning of the reaction, the degradation solution (containing 100 mmol / L ascorbic acid, 10 mmol / L CuSO4·5H2O, and 10 mmol / L FeSO4·7H2O) is added in two batches, each time for 1 hour, and the reaction is carried out for 2 hours. The reaction solution was ultrafiltered using a 300 kDa ultrafiltration membrane (Millipore), and the permeate was collected. The permeate was then concentrated and replaced using a 10 kDa ultrafiltration membrane (Millipore) with 0.9% NaCl solution and pure water as replacement solutions, with each replacement performed three times at 10 times its original volume. The retentate after replacement was collected, filtered through a 0.45 μm filter, and then lyophilized.

[0157] Example 2 Preparation of Carrier Protein

[0158] The carrier protein rPly (MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTACNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILCGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREATGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND, SEQ ID NO: 1) was prepared according to the method disclosed in Patent CN113480622A;

[0159] rTTc (KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYDTNEYSIISSMKKYSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLSDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVAYSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYSGLKFIIKRYTPNNEIDSFVRSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKDASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKTLTCDWYFVPTDEGWTND, SEQ ID NO: 2) was prepared according to the method disclosed in Patent CN119930770A;

[0160] rCRM197 (SEQ ID NO: 3) was recombinantly expressed in Escherichia coli. The bacterial cells were homogenized to obtain a lysate, which was then clarified by passing the lysate through a 0.2 μm membrane to obtain a clear permeate. The permeate was then concentrated and replaced by passing the permeate through a 10 kDa membrane to obtain a crude sample. The crude sample was purified by DEAEFF, Phenyl HP and Heparin FF column chromatography. Alternatively, it can be purchased (Beijing Bolede Biotechnology Co., Ltd.).

[0161] rPhtD (SEQ ID NO: 4) was recombinantly expressed in E. coli. The bacterial cells were homogenized to obtain a lysate, which was then clarified by passing the lysate through a 0.2 μm membrane to obtain a clear permeate. The permeate was then concentrated and replaced by passing the permeate through a 10 kDa membrane to obtain a crude sample. The crude sample was purified by DEAE FF and Phenyl HP column chromatography.

[0162] Example 3: Preparation of capsular polysaccharide conjugates of 31 pneumococcal serotypes using rPly as the carrier protein

[0163] The hydrolyzed capsular polysaccharide / capsular polysaccharide (0.1 g) obtained in Example 1 and the rPly (0.1 g) obtained in Example 2 were conjugated using the following method to obtain 31 monovalent conjugate stock solutions.

[0164] Purified polysaccharides or hydrolyzed sugars are chemically activated to enable them to react with carrier proteins. The purified polysaccharides can be ligated to adapters; once activated or ligated, each capsular polysaccharide conjugates with a carrier protein to form a glycoconjugate. Polysaccharide conjugates can be prepared using known coupling techniques. For example, dissolving the polysaccharide in NaCl solution, adjusting the pH to 8.45–9.05 after complete dissolution, adding dissolved CDAP solution, followed by triethylamine solution to maintain pH 8.30–9.20, reacting for 2 min, and immediately adding the carrier protein to the reaction beaker after 2 min, reacting for 6–24 h; after the reaction, diluting to 10 L with NaCl solution, concentrating and replacing four times using a 300 kDa ultrafiltration membrane (Millipore), finally collecting the retentate, and filtering through a 0.22 μm syringe filter to obtain the final product.

[0165] The detection results of the original solutions of various pneumococcal polysaccharide-rPly protein conjugates are shown in Table 1.

[0166] Table 1. Detection results of stock solutions of polysaccharide-rPly protein conjugates from various types of pneumococcus

[0167]

[0168] Note: The molecular sizes in the table above were determined by SEC-MALS-RI.

[0169] Example 4: Preparation of capsular polysaccharide conjugates of 31 pneumococcal serotypes using rCRM197 as the carrier protein

[0170] The hydrolyzed capsular polysaccharide / capsular polysaccharide (0.1g) obtained in Example 1 and rCRM197 (0.1g) obtained in Example 2 were conjugated using the method described in Example 3 to obtain 31 monovalent conjugate stock solutions.

[0171] The detection results of the original solutions of various pneumococcal polysaccharide-rCRM197 protein conjugates are shown in Table 2.

[0172] Table 2. Detection results of stock solutions of polysaccharide-rCRM197 protein conjugates from different types of pneumococci.

[0173]

[0174] Note: The molecular sizes in the table above were determined by SEC-MALS-RI.

[0175] Example 5: Preparation of capsular polysaccharide conjugates of 31 pneumococcal serotypes using rTTc as the carrier protein

[0176] The hydrolyzed capsular polysaccharide / capsular polysaccharide (0.1 g) obtained in Example 1 and the rTTc (0.1 g) obtained in Example 2 were conjugated using the method described in Example 3 to obtain 31 monovalent conjugate stock solutions.

[0177] The detection results of the original solutions of various pneumococcal polysaccharide-rTTc protein conjugates are shown in Table 3.

[0178] Table 3. Detection results of stock solutions of polysaccharide-rTTc protein conjugates from different types of pneumococcus

[0179]

[0180] Note: The molecular sizes in the table above were determined by SEC-MALS-RI.

[0181] Example 6: Preparation of capsular polysaccharide conjugates of 11 pneumococcal serotypes using rPhtD as the carrier protein.

[0182] The hydrolyzed capsular polysaccharides obtained in Example 1 (each hydrolyzed capsular polysaccharide has an equal mass of 0.1 g) were conjugated with rPhtD (0.1 g) obtained in Example 2 using the method described in Example 3 to obtain 11 monovalent conjugate stock solutions.

[0183] The detection results of the original solutions of various pneumococcal polysaccharide-rPhtD protein conjugates are shown in Table 4.

[0184] Table 4. Detection results of stock solutions of polysaccharide-rPhtD protein conjugates from various types of pneumococci.

[0185]

[0186] Example 7 Immunogenicity Study of Monovalent Conjugates of Different Carrier Proteins

[0187] This study used conjugate stock solutions prepared with different carrier proteins as antigens to compare the changes in IgG content in different types of pneumococcal polysaccharide conjugate stock solutions with different protein carriers, aiming to determine the best carrier protein for each type of polysaccharide antigen.

[0188] This study used BALB / c mice as an animal model. A three-dose immunization program was used (two weeks apart, with 0.05 mL of the monovalent conjugate stock solution obtained in Examples 3-6 injected into the leg muscle for immunization, and the blank control group injected with the same volume of PBS). Different doses were immunized at two-week intervals. Blood samples were collected from the eyeballs two weeks after the second and third doses of immunization (three independent experiments, n=6 in each group). The serum content of capsular polysaccharide-specific IgG was measured by indirect ELISA.

[0189] Polysaccharides were serially diluted to serve as the coating antigen. The protein coating concentration was 10 μg / mL. 100 μl of the protein was added to each well of an ELISA plate (Corning 42592), and the plate was incubated overnight (16-18 h) at 2-8°C, followed by one wash. 200 μl of 1x coating buffer containing 1% BSA was added to each well, and the plate was incubated at 37°C for 1 h, followed by 5 washes. 200 μl of serum was added to each well in row A, followed by a 2-fold serial dilution (serum diluent was 1× antibody diluent TBS or 10 mM PB, pH 7.0, containing 0.9% NaCl and 0.5% BSA). The plate was sealed and incubated at 37°C for 2 h, followed by 5 washes. 100 μl of 4000-fold diluted alkaline phosphatase-labeled goat anti-mouse IgG (SouthernBiotech, secondary antibody diluent was 1× antibody diluent TBS) was added to each well, and the plate was incubated at 37°C for 1 h, followed by 5 washes. Add 100 μl of substrate dissolution buffer (pNPP or BCIP / NBT) to each well and incubate at 37°C in the dark for 1.5 h. Add 50 μl of 3M NaOH stop solution to each well and read the values ​​of the ELISA plate using a microplate reader. The cutoff value was determined to be 0.2 based on the negative serum source (unimmunized mice as blank control).

[0190] The selection criteria for optimal carrier proteins for each type of capsular polysaccharide were as follows: Criterion 1: Firstly, carrier proteins that maintained a high geometric mean concentration (GMC) level after both 2 and 3 injections were preferred; secondly, if the GMC trends after 2 and 3 injections were inconsistent: if the GMC values ​​of different carriers differed significantly after 3 injections, the results of 3 injections were used as the primary criterion; if there was no significant difference in GMC values ​​of different carriers after 3 injections, the comprehensive criterion was that the number of types corresponding to each carrier did not exceed 13; Criterion 2: Selection was based on the results of 2 injections; Criterion 3: Selection was based on the results of 3 injections. The results are shown in Table 5, and the optimal combinations obtained are groups based on criteria 1, 2, and 3 in Table 6.

[0191] Table 5. Immunogenicity evaluation results of conjugate stock solutions with different carriers

[0192]

[0193]

[0194]

[0195] Note: All significance analyses in the table are within-dosage comparisons; in the significance analysis, uppercase letters (A, B...) indicate significance at the P < 0.01 level, and lowercase letters (a, b...) indicate significance at the P < 0.05 level.

[0196] Table 6. Compatibility Combinations of Different Types with Different Carrier Proteins

[0197]

[0198] Based on the above selection criteria and to avoid vector inhibition caused by an excessive number of one type of vector, each vector was designed to be compatible with approximately 10 types. As shown in Table 5, the optimal carrier protein for the 6 capsular polysaccharide types is rTTc. To meet the requirement of rTTc carrying 10 capsular polysaccharide types, 4 types with no significant difference from other vectors were selected, thus satisfying the requirement of 10 types. For other vectors, based on the results obtained from criteria two and three, capsular polysaccharides exceeding 10 types were adjusted to approximately 10 types each for rPly and rCRM197 vectors, based on the lack of significant difference in immunogenicity; this resulted in criteria four and five groups.

[0199] Based on the above combination results and the published clinical results of marketed products, the final optimal combination groups are adjusted as shown in Table 7 below.

[0200] Table 7. Adjusted compatibility combinations of each type and carrier protein

[0201]

[0202] Example 8: Immunogenicity Study of 31-valent Pneumococcal Conjugate Vaccines with Different Vectors

[0203] This study used the 23-valent pneumococcal polysaccharide vaccine Huiyikang (PPV23*) as a control to compare several 31-valent pneumococcal conjugate vaccines: 1) a 31-valent pneumococcal conjugate vaccine composition (PCV31-rPly) formulated from a single-vector (rPly) conjugate stock solution; 2) a 31-valent pneumococcal conjugate vaccine (PCV31-rCRM197) formulated from a single-vector (rCRM197) conjugate stock solution; 3) a 31-valent pneumococcal conjugate vaccine (PCV31-rTTc) formulated from a single-vector (rTTc) conjugate stock solution; and 4) a 31-valent pneumococcal conjugate vaccine (PCV31-rTTc) formulated from a tri-vector (rPly) conjugate stock solution. This study focuses on pneumococcal conjugate vaccines formulated with three vectors (rPly, rCRM197, and rTTc) (component 1); three vectors (rPly, rCRM197, and rTTc) (component 2); and three vectors (rPly, rCRM197, and rTTc) (component 3). The aim of this study is to investigate the differences in immunogenicity between the two types of conjugate vaccines formulated with three vectors and single vectors, and to screen for the most effective combination from the three-vector combinations.

[0204] The formulation of the pneumococcal conjugate vaccine in this study is as follows:

[0205] In each 0.5 ml of vaccine, the content of polysaccharides of each type (excluding type 6B) is set at 2 μg, but the content of polysaccharides of serotype 6B is 4 μg; the content of polysorbate 80 is 100 μg, the content of sodium chloride is 4.5 mg, and the dosage of aluminum phosphate adjuvant is 0.125 mg.

[0206] Using BALB / c mice as an animal model, 10 mice per group were administered a two-dose immunization program (two weeks apart). 0.05 mL of immunized blood (1 / 10 of the human dose) was injected intramuscularly into the leg. Blood samples were collected from the orbital rim two weeks after the first and second immunizations. The titers of antigen-specific IgG were detected using an indirect ELISA method, the same as in Example 7.

[0207] The immunization groups are shown in Table 8.

[0208] Table 8 Immunogen Information

[0209]

[0210] Note: Control group: *PPV23 is a 23-valent pneumococcal polysaccharide vaccine (commercially available under the name "Huiyikang", abbreviated as PPV23 (Huiyikang)), which includes types 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F; the 31-valent pneumococcal polysaccharide conjugate vaccine (PCV31-rPly, PCV31-rCRM197, PCV31-rTTc, combination 1, combination 2 and combination 3) adds types 6A, 15A, 15C, 16F, 23A, 23B, 31 and 35B to the polysaccharide types in addition to the 23-valent vaccine.

[0211] The results are shown in Table 9: (1) After 1 and 2 doses of immunization, the antibody levels of the polysaccharide conjugate vaccine were higher or significantly higher than those of the control group PPV23; (2) Compared with the single-vector and triple-vector PCV31 immunization, after 1 dose of immunization, most types (19 types) of PCV31 in the triple-vector showed an advantage, and the antibody level of type 8 showed a significant advantage. After 2 doses of immunization, most types (26 types) showed an advantage, and types 10A, 16F, 17F and 19F showed a significant or extremely significant advantage; considering all factors, the immunogenicity of the finished triple-vector combination in mice was better than that of the single-vector combination.

[0212] Table 9. Immunogenicity studies of 31-valent pneumococcal conjugate vaccines with different vectors.

[0213]

[0214]

[0215]

[0216]

[0217] Note: "N / A" in the table indicates that this item is not applicable; in the significance analysis, uppercase letters (A, B...) indicate significance at the P < 0.01 level, and lowercase letters (a, b...) indicate significance at the P < 0.05 level.

[0218] Table 10 shows the results of three-carrier combinations 1, 2, and 3. After one immunization, the levels of specific antibodies against each polysaccharide type were higher in combination 1 than in combinations 2 and 3, especially for types 1, 2, 9V, 11A, and 14, where significant or highly significant differences were observed. After two immunizations, the levels of specific antibodies against each polysaccharide type remained higher in combination 1 than in combinations 2 and 3, especially for types 3 and 14, where significant or highly significant differences were observed. In this study, three-carrier combination 1 showed greater advantages compared to the other two combinations.

[0219] Table 10 Immunogenicity studies of 31-valent pneumococcal conjugate vaccines with different tri-vector combinations

[0220]

[0221]

[0222]

[0223] Note: "N / A" in the table indicates that this item is not applicable; all significance analyses in the table are within-dosage comparisons; in the significance analysis, uppercase letters (A, B...) indicate significance at the P < 0.01 level, and lowercase letters (a, b...) indicate significance at the P < 0.05 level.

[0224] Example 9: Study on the ratio of antigen and aluminum phosphate adjuvant in a three-vector pneumococcal conjugate vaccine

[0225] This study formulated 31 monovalent stock solutions containing three different carriers into active ingredients at specific concentrations (component 2), which were then combined with aluminum phosphate adjuvants at specific concentrations to prepare pneumococcal conjugate vaccines at different doses. The adjuvant dose of 0.125 mg / dose corresponded to two antigen dose groups: 2 μg / dose and 4 μg / dose (except for 6B, which were 4 μg / dose and 8 μg / dose). The dose without adjuvant corresponded to two antigen dose groups: 3 μg / dose and 4 μg / dose (except for 6B, which were 6 μg / dose and 8 μg / dose).

[0226] The pneumococcal conjugate vaccine formulations used in this study are as follows: For the adjuvant-containing formulation, each 0.5 ml of vaccine contains 2 μg / 4 μg of polysaccharides for each type (excluding 6B), but 4 μg / 8 μg for serotype 6B; 100 μg of polysorbate 80, 4.5 mg of sodium chloride, and 0.125 mg of aluminum phosphate adjuvant. For the adjuvant-free formulation, each 0.5 ml of vaccine contains 3 μg / 4 μg of polysaccharides for each type (excluding 6B), but 6 μg / 8 μg for serotype 6B; 100 μg of polysorbate 80, 4.5 mg of sodium chloride, and 1.23 mg of sodium acetate.

[0227] Using BALB / c mice as an animal model, 10 mice per group were administered a two-dose immunization program (two weeks apart). 0.05 mL of immunized blood (1 / 10 of the human dose) was injected intramuscularly into the leg. Blood samples were collected from the orbital rim two weeks after the first and second immunizations. The titers of antigen-specific IgG were detected using an indirect ELISA method, the same as in Example 7.

[0228] The results are shown in Table 11. After immunization with the three-vector PCV31, there was no significant difference in immunogenicity between the aluminum phosphate adjuvant and the unadjuvanted types in most categories. The adjuvant dosage and antigen amount had different effects on immunogenicity, all showing good immunogenicity. Extremely high immune response levels could also be induced at the unadjuvanted immunization dose, further improving the safety of the vaccine.

[0229] Table 11 Study on the ratio of antigen and aluminum phosphate adjuvant in tri-carrier pneumococcal conjugate vaccines

[0230]

[0231]

[0232]

[0233] Note: "N / A" in the table indicates that this item is not applicable; all significance analyses in the table are within-dosage comparisons; in the significance analysis, uppercase letters (A, B...) indicate significance at the P < 0.01 level, and lowercase letters (a, b...) indicate significance at the P < 0.05 level.

[0234] Example 10 Study on different antigen contents of a three-vector pneumococcal conjugate vaccine

[0235] In this study, 31 monovalent stock solutions containing three carriers were formulated into active ingredients of a certain concentration (component 1), and then combined with excipients to formulate pneumococcal conjugate vaccines with different antigen doses.

[0236] The pneumococcal conjugate vaccine formulation for this study is as follows: In each 0.5 ml of vaccine, the antigen content of each experimental group is shown in Table 12, the content of polysorbate 80 is 100 μg, the content of sodium chloride is 4.5 mg, and the content of sodium acetate is 1.23 mg.

[0237] Table 12 Anchoring Type Test Design

[0238]

[0239] Using New Zealand white rabbits as the research subject, rabbits were administered a single intramuscular injection of 0.5 ml of human dose. A three-dose immunization program was followed, with immunizations administered two weeks apart. Blood samples were collected from the eyes two weeks after the first, second, and third immunizations. The titers of antigen-specific IgG were detected using an indirect ELISA method, as described in Example 7. The functional activity of specific antibodies in serum of types 1, 3, 5, 6A, 6B, 7F, 10A, 14, 15B, 19A, 19F, and 33F was quantitatively determined using a standard opsonization phagocytosis activity assay (OPA) to evaluate their actual protective efficacy in mediating immune cell phagocytosis and clearance of pneumococcus.

[0240] The results are shown in Tables 13, 14, and 15. In this study, in a rabbit model, the immune response levels of pneumococcal polysaccharides 1, 3, 6B, 19A, and 19F significantly increased with increasing antigen dose, exhibiting a dose-dependent increase in GMC (geometric mean concentration), indicating that increasing the dose effectively enhances the immunogenicity of these types. The GMC levels of more than half of the other types also increased with increasing antigen dose. In both single-dose and two-dose immunization programs, the 10 μg dose group had 16 types with a GMC ratio (compared to the 4 μg dose group as a control) greater than 1.5, significantly more than other dose groups. The trend of functional antibody titer changes was consistent with the trend of IgG content changes. Based on the above dose-response relationship, this study selected the 10 μg dose group as the optimal dose of the polysaccharide antigen.

[0241] Table 13 Results of IgG GMC in antiserum before immunization and after different doses of immunization in different antigen dosage groups

[0242]

[0243]

[0244]

[0245] Table 14 Comparison of GMC ratios for different antigen dosage groups and injection sessions (with the 4μg dosage group as the control)

[0246]

[0247] Table 15. Results of OPA functional antibody titers in antiserum before immunization and after different doses of immunization in different antigen dosage groups.

[0248]

[0249]

[0250] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A composition of 31-valent pneumococcal capsular polysaccharide, characterized in that, The composition contains 31 types of pneumococci. The serological capsular polysaccharides are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B. Among them, the content of capsular polysaccharides of individual types 1, 3, 6B, 19A, and 19F in the composition is higher than that of other types.

2. The composition according to claim 1, characterized in that, The composition is divided into the following three groups based on the content of individual capsular polysaccharides: Group 1): Capsular polysaccharides of types 1, 3, 6B, 19A, and 19F; 2) Groups: Capsular polysaccharides of types 15A, 15C, 16F, 23A, 23B, 31, and 35B; and, 3) Groups: Capsular polysaccharides of types 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F; among them, the content of capsular polysaccharides of different serotypes within the same group is the same, while the content of capsular polysaccharides of different types varies between groups; Preferably, for a single type of capsular polysaccharide in the composition, the capsular polysaccharide content in group 1) is 2-6 times, for example 4-5 times, that in group 2); and / or, the capsular polysaccharide content in group 3) is 1-3 times, for example 2 times, that in group 2).

3. The composition according to claim 2, characterized in that, The composition satisfies one or more of the following conditions: a. 1) The content of a single capsular polysaccharide in the group is 4~10 μg / 0.5 mL; for example, 10 μg / 0.5 mL, 8 μg / 0.5 mL, 6 μg / 0.5 mL or 4 μg / 0.5 mL; b. 2) The content of single-type capsular polysaccharides in group 1.5~2.5μg / 0.5mL; and, c. 3) The content of single-type capsular polysaccharides in group 3.5~4.5μg / 0.5mL; Preferably, in the composition, the content of a single type of capsular polysaccharide in group 1) is 8-10 μg / 0.5 mL, the content of a single type of capsular polysaccharide in group 2) is 2 μg / 0.5 mL, and the content of a single type of capsular polysaccharide in group 3) is 4 μg / 0.5 mL.

4. The composition according to any one of claims 1-3, characterized in that, The composition further comprises a carrier protein, wherein the capsular polysaccharide forms a conjugate with the carrier protein; and / or, the composition does not contain an adjuvant; Preferably, the carrier protein is selected from one or more of pneumococcal hemolysin, tetanus toxoid, diphtheria toxin non-toxic mutant, and recombinant pneumococcal histidine triplet protein; the tetanus toxoid is preferably the tetanus toxin C fragment; More preferably, the carrier protein includes pneumococcal hemolysin and / or tetanus toxin C fragment; it also includes one or more of tetanus toxoid, a non-toxic mutant of diphtheria toxin, and recombinant pneumococcal histidine triplet protein; the carrier protein preferably includes pneumococcal hemolysin, a tetanus toxin C fragment, and a non-toxic mutant of diphtheria toxin. More preferably, in the composition: 1) One or more capsular polysaccharides from 31 serotypes are conjugated with pneumococcal hemolysin; 2) One or more capsular polysaccharides from the 31 serotypes were conjugated to a non-toxic mutant of diphtheria toxin; or, 3) One or more capsular polysaccharides from the 31 serotypes were conjugated to the tetanus toxin C fragment.

5. The composition according to claim 4, characterized in that, The composition is divided into three groups based on the different carrier proteins that bind to the capsular polysaccharide: i) Group: Contains one or more of the capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 11A, 14, 17F, 19A, 19F, 20, 22F, 23B, 23F, 31 and 35B; preferably contains 9 to 13 types, such as 10, 11 or 13 types; Group ii): comprising one or more of serotypes 1, 2, 5, 7F, 8, 9V, 9N, 10A, 11A, 14, 15A, 15B, 15C, 16F, 18C, 20, 31, and 33F capsular polysaccharides; preferably comprising 10-13, for example 11 or 12; and, iii) Group: Contains one or more capsular polysaccharides from serotypes 3, 5, 7F, 8, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 22F, 23A, and 23B; preferably containing 5-11 types, such as 6, 9, or 10 types; wherein, within the same group, the carrier protein bound to the single-type capsular polysaccharide is the same, and between different groups, the carrier protein bound to the single-type capsular polysaccharide is different; and the single-type capsular polysaccharide appears only once in the three groups; Preferably, the composition satisfies one or more of the following conditions: In group i), the capsular polysaccharides were conjugated with pneumococcal hemolysin, respectively. (ii) The capsular polysaccharides in group ii) were conjugated with a non-toxic mutant of diphtheria toxin; and, (iii) The capsular polysaccharides in group iii) were conjugated to the tetanus toxin C fragment, respectively; More preferably, the composition comprises: 1) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23B, 23F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 18C, 31, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 12F, 22F, and 23A with the C fragment of tetanus toxin. 2): Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 20, 23B, 23F, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 18C, 19A, 20, 23A, and 31 with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, and 14 with tetanus toxin C fragment; 3): Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23A, 23B, and 23F with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 11A, 15A, 15B, 15C, 16F, 18C, 31, 33F, and 35B with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, and 22F with the C fragment of tetanus toxin. 4): Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 23B, 23F, 31, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 11A, 15B, 15C, 16F, and 18C with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment; 5): Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6A, 6B, 14, 17F, 19F, 23B, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 9V, 9N, 11A, 15B, 15C, 16F, 18C, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment; 6): Conjugates formed by conjugating capsular polysaccharides of serotypes 4, 6A, 6B, 11A, 17F, 19F, 22F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 5, 7F, 8, 14, 15A, 15C, 16F, 20, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 3, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 23A, and 23B with tetanus toxin C fragment; or, 7): Conjugates formed by conjugating capsular polysaccharides of serotypes 3, 4, 6A, 6B, 11A, 14, 17F, 19F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 9V, 10A, 15A, 15B, 15C, 16F, 18C, 20, and 33F with non-toxic mutants of diphtheria toxin; and conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 9N, 12F, 19A, 22F, 23A, and 23B with tetanus toxin C fragment.

6. The composition according to claim 4 or 5, characterized in that, The composition satisfies one or more of the following conditions: 1) The capsular polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B are hydrolyzed capsular polysaccharides; 2) The pneumococcal hemolysin has the amino acid sequence shown in SEQ ID NO: 1; 3) The tetanus toxin C fragment has the amino acid sequence shown in SEQ ID NO: 2; 4) The diphtheria toxin non-toxic mutant has the amino acid sequence shown in SEQ ID NO: 3; and, 5) The ratio of capsular polysaccharide to carrier protein in the composition is (0.5-1.5):

1.

7. A vaccine, characterized in that, The vaccine comprises the composition as described in any one of claims 1-6, the vaccine is used to induce an immune response in the body against pneumococcus, and the vaccine further comprises excipients; Preferably, the excipients comprise one or more of the following: sodium chloride, sodium acetate, citric acid, polysorbate 80, trehalose, sucrose, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and arginine. More preferably, the vaccine meets one or more of the following criteria: 1) The sodium chloride content in the vaccine is 3.75~4.75mg / 0.5mL, for example 4.5mg / 0.5mL; 2) The sodium acetate content in the vaccine is 1~3 mg / 0.5 mL, for example 1.23 mg / 0.5 mL; and, 3) The content of polysorbate 80 in the vaccine is 80~2500μg / 0.5mL, for example 100μg / 0.5mL.

8. The vaccine as described in claim 7, characterized in that, The vaccine does not contain adjuvants.

9. The vaccine as described in claim 7 or 8, characterized in that, The vaccine contains 8-10 μg / 0.5 mL of monotype capsular polysaccharides of types 1, 3, 6B, 19A, and 19F; 2 μg / 0.5 mL of monotype capsular polysaccharides of types 15A, 15C, 16F, 23A, 23B, 31, and 35B; and 4 μg / 0.5 mL of monotype capsular polysaccharides of types 2, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F, and 33F. Preferably, the vaccine contains: 1) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23B, 23F, and 35B with pneumococcal hemolysin; 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 18C, 31, and 33F with non-toxic mutants of diphtheria toxin; 3) Conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 12F, 22F, and 23A with the C fragment of tetanus toxin. 2) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 20, 23B, 23F, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 18C, 19A, 20, 23A, and 31 with diphtheria toxin non-toxic mutants; and conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, and 14 with tetanus toxin C fragment. 3) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 4, 6A, 6B, 14, 17F, 19A, 19F, 20, 23A, 23B, and 23F with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 9V, 9N, 11A, 15A, 15B, 15C, 16F, 18C, 31, 33F, and 35B with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, and 22F with the C fragment of tetanus toxin. 4) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6B, 14, 17F, 19F, 23B, 23F, 31, 33F, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 6A, 9V, 9N, 11A, 15B, 15C, 16F, and 18C with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment; 5) Conjugates formed by conjugating capsular polysaccharides of serotypes 1, 3, 6A, 6B, 14, 17F, 19F, 23B, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 2, 4, 9V, 9N, 11A, 15B, 15C, 16F, 18C, and 33F with diphtheria toxin non-toxic mutants; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 10A, 12F, 15A, 19A, 20, 22F, and 23A with tetanus toxin C fragment; 6) Conjugates formed by conjugating capsular polysaccharides of serotypes 4, 6A, 6B, 11A, 17F, 19F, 22F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 5, 7F, 8, 14, 15A, 15C, 16F, 20, and 33F with a non-toxic mutant of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 3, 9N, 9V, 10A, 12F, 15B, 18C, 19A, 23A, and 23B with the C fragment of tetanus toxin; or, 7) Conjugates formed by conjugating capsular polysaccharides of serotypes 3, 4, 6A, 6B, 11A, 14, 17F, 19F, 23F, 31, and 35B with pneumococcal hemolysin; conjugates formed by conjugating capsular polysaccharides of serotypes 1, 2, 9V, 10A, 15A, 15B, 15C, 16F, 18C, 20, and 33F with non-toxic mutants of diphtheria toxin; conjugates formed by conjugating capsular polysaccharides of serotypes 5, 7F, 8, 9N, 12F, 19A, 22F, 23A, and 23B with tetanus toxin C fragment; More preferably, the vaccine meets one or more of the following criteria: 1) The capsular polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 22F, 23A, 23B, 23F, 31, 33F, and 35B are hydrolyzed capsular polysaccharides; 2) The pneumococcal hemolysin has the amino acid sequence shown in SEQ ID NO: 1; 3) The tetanus toxin C fragment has the amino acid sequence shown in SEQ ID NO: 2; 4) The diphtheria toxin non-toxic mutant has the amino acid sequence shown in SEQ ID NO: 3; and, 5) The ratio of capsular polysaccharide to carrier protein in the composition is (0.5-1.5):

1.

10. Use of the composition according to any one of claims 1-6 in the preparation of a medicament for the prevention of pneumococcal-related diseases; preferably, the medicament is a vaccine.

11. A method for preparing the composition according to any one of claims 1-6, characterized in that, The method includes the following steps: 1) Capsular polysaccharides of each serotype were conjugated with carrier proteins to obtain conjugates; and, 2) Mix the conjugates obtained in step 1) to obtain a composition; Preferably, in step 1), the capsular polysaccharide is activated and then conjugated to the carrier protein. The activation is chemical activation, such as cyanogen bromide method, CDAP method or periodic acid oxidation method. Preferably, a hydrolysis step is also included before the activation of the capsular polysaccharide, such as acid hydrolysis, hot water hydrolysis or enzymatic hydrolysis.

12. A method for preparing a pneumococcal vaccine, characterized in that, The method includes the step of adding excipients to the composition as described in any one of claims 1-6; Preferably, the excipient is as defined in the vaccine as described in any one of claims 7-9; and / or, the method further includes a sterilization step, such as sterilization by filtration.

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