WO2023025002A1 - Streptococcus pneumoniae conjugate vaccine composition - Google Patents

Abstract

The present invention provides a glycoconjugate and an immunogenic composition comprising the glycoconjugate, and also provides a preparation method for the glycoconjugate, which comprises: oxidizing a primary hydroxyl group of bacterial capsular polysaccharide, which is conjugated to a carrier protein directly or by passing through a spacer group, so as to prepare and obtain a glycoconjugate. Also disclosed are an application of the glycoconjugate and immunogenic composition in the preparation of a drug or vaccine to prevent and/or treat individual Streptococcus pneumoniae infections and diseases related to Streptococcus pneumoniae.

Description

A pneumococcal conjugate vaccine composition technical field

The invention relates to the technical field of vaccine development, in particular to a glycoconjugate prepared by reacting capsular polysaccharide of serotype Streptococcus pneumoniae with a carrier protein, an immunogenic composition comprising the glycoconjugate, and the Application of the above-mentioned glycoconjugates and immunogenic compositions in the preparation of drugs or vaccines for preventing and/or treating individual Streptococcus pneumoniae infection and diseases related to Streptococcus pneumoniae.

Background technique

The invention relates to the field of medical biotechnology development, in particular to the preparation of a multivalent pneumococcal vaccine composition, which consists of 24 serotypes, including pneumococcal serotypes 1, 2, 3, 4, 5, and 6A , 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F for prophylaxis against pneumococci of the included serotypes infection.

Pneumococcus (Streptococcus pneumoniae) is a Gram-positive bacterium, and the capsular polysaccharide outside the cell wall has a thicker capsule. Pneumococcus is the main pathogen that causes pneumonia, bacteremia, and meningitis in children and the elderly. Both have high morbidity and mortality. The pathogenicity and serotype of bacteria are often related to the composition and structure of the capsular polysaccharide. So far, more than 90 different serotypes of the capsular polysaccharide of Streptococcus pneumoniae have been found, and more than 20 of them have been used to prepare bacteria caused by Streptococcus pneumoniae infection. Disease vaccine. Streptococcus pneumoniae conjugate vaccine (PCV) is a pneumococcal vaccine used to prevent diseases caused by Streptococcus pneumoniae. Pfizer's thirteen-valent vaccine Prevenar 13 is currently available worldwide.

According to the serotypes of pneumococci prevalent in China, the inventors developed a 24-valent pneumococcal conjugate vaccine, including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.

Among them, the traditional sodium periodate method or CDAP (1-cyano-4-(dimethylamino)pyridine tetrafluoroborate) method can be used for various serotypes in the research process to obtain highly immunogenic conjugates. However, the stability of activated polysaccharides obtained by sodium periodate method or CDAP method for 12F serotype polysaccharides is poor, and the immune response of the final conjugate is weak.

Patent CN104870463A discloses a brand-new method for preparing conjugates from 12F serotype capsular polysaccharide, developed by Pfizer. Specifically, methods for preparing glycoconjugates comprising saccharides conjugated to carrier proteins, immunogenic compositions comprising such glycoconjugates, and the use of stable nitroxyl-related reagents/oxidants as oxidizing species Methods of such glycoconjugates and immunogenic compositions. The method uses TEMPO-NCS to selectively oxidize primary hydroxyl groups to generate aldehyde groups to prepare activated polysaccharides.

The non-patent literature TEMPO-mediated glycoconjugation: a scheme for the controlled synthesis of polysaccharide conjugates (Carbohydrate Research 346 (2011) 343–347) discloses a method: using TEMPO-NaClO to selectively oxidize the polysaccharide on the capsular polysaccharide structure of Gram-negative bacteria The primary hydroxyl group is a carboxylic acid, and then the carboxylic acid is combined with the carrier protein, but this method is difficult to control, and the oxidation rate of NaClO is too fast.

Non-patent literature Effect of temperature modulations on TEMPO-mediated Regioselective oxidation of unprotected carbohydrates and nucleosides discloses a method: use TEMPO-iodobenzenediacetic acid to selectively oxidize primary hydroxyl groups to carboxylic acids, but control the amount of iodobenzenediacetic acid and no In the presence of a base, the primary hydroxyl group can only be oxidized to the aldehyde group, and no longer continue to be oxidized to the carboxylic acid.

Patent CN111821432 discloses a method, using sodium periodate to oxidize capsular polysaccharides to obtain activated polysaccharides, then derivatized by spacers, and finally combined with carrier protein, but the stability of the activated polysaccharides obtained by this method is poor, and the immune response of the conjugates is weak Weak, unable to meet the demand for vaccines.

Contents of the invention

According to the technical defects or technical barriers of the above methods, the present invention provides a variety of methods for preparing 12F type pneumococcal capsular polysaccharides and protein conjugates, using different activation (reaction) site reactions of polysaccharides to obtain 12F type pneumococcal capsules The glycoconjugates of polysaccharides and protein carriers are coupled, and the activation sites of 12F type pneumococcal capsular polysaccharides are disclosed. The activation sites are: acetylaminogalactosyl primary hydroxyl group (β-D -GalpNAc), galactopyranosyl primary hydroxyl group (α-D-Galp), or glucopyranosyl primary hydroxyl group (α-D-Glcp).

The present invention also provides a method for the preparation of glycoconjugates, which is applicable to any polysaccharide containing primary hydroxyl groups, and has obvious advantages over sodium periodate or CDAP activation methods. The sodium periodate oxidation method is mainly used in After the sugar ring with adjacent hydroxyl groups is activated and the sugar ring structure is destroyed, the 12F serotype polysaccharide is very unstable.

The oxidation method employed in the present invention requires only that the capsular polysaccharide contain primary hydroxyl groups. The present invention further provides an immunogenic composition comprising said glycoconjugate. The present invention also provides the application of the glycoconjugate and the immunogenic composition in the preparation of drugs or vaccines for preventing and/or treating individual Streptococcus pneumoniae infection and diseases related to Streptococcus pneumoniae.

The conjugate of the present invention has high stability, high immunogenicity, and significant bactericidal effect compared with the conjugate obtained by using periodate activation, CDAP activation and carrier protein in the prior art and the TEMPO-NCS method enhanced.

Specifically, the first aspect of the present invention provides a glycoconjugate obtained by oxidizing the primary hydroxyl group of a bacterial capsular polysaccharide and coupling it to a carrier protein directly or via a spacer group .

Preferably, the bacterial capsular polysaccharide is selected from Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C , 19A, 19F, 20, 22F, 23F or 33F type capsular polysaccharide, further preferably, the bacterial capsular polysaccharide is Streptococcus pneumoniae type 12F capsular polysaccharide.

Preferably, the bacterial capsular polysaccharide activation site includes β-D-GalpNAc, α-D-Galp or α-D-Glcp.

Preferably, the Streptococcus pneumoniae 12F capsular polysaccharide can be natural or synthetic.

Preferably, the primary hydroxyl group in the bacterial capsular polysaccharide is oxidized with a nitroxyl compound and an iodine oxidizing agent, further preferably, the nitroxyl compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical or a compound containing 2,2,6,6-tetramethyl-1-piperidinyloxy radical structure, more preferably, the iodine oxidizing agent is PIA.

Preferably, compounds containing 2,2,6,6-tetramethyl-1-piperidinyloxy radical structure include but not limited to 2,2,6,6-tetramethylpiperidinium oxide, 2,2 ,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinyloxy, 4-phosphonyloxy-TEMPO, 4-oxo-TEMPO, 4-isothiocyanato -TEMPO, 4-(2-iodoacetamido)-TEMPO radical, 4-hydroxy-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl, 4-cyano -TEMPO, 4-methoxy-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO or 4-amino-TEMPO.

Preferably, the carrier protein contains one or more amino or carboxyl groups. The carrier protein can be a related protein antigen from the target pathogen that enhances the specific immune response to the pathogen, or a general immunogenic protein that mainly acts as an adjuvant or general immune response stimulator.

Further preferably, the carrier protein is selected from diphtheria toxoid mutant (CRM197/CRM), tetanus toxoid (TT), outer membrane protein from Gram-negative bacteria, Haemophilus influenzae surface lipoprotein, Fusion protein formed by Haemophilus influenzae HiD protein gene and Haemophilus influenzae Hin47 protein gene in a 1:1 manner, pertussis toxin, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein or respiratory syncytia Viral F and G proteins or active parts thereof.

In a specific embodiment of the present invention, the carrier protein is CRM197 or TT.

Preferably, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3 (for example, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3).

Further preferably, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8-2.

In a specific embodiment of the present invention, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 1.0 to 1.3.

In a specific embodiment of the present invention, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8 to 1.2.

Preferably, for every 10 to 30 sugar repeating units of the bacterial capsular polysaccharide, there is at least one covalent bond between the carrier protein and the bacterial capsular polysaccharide.

The second aspect of the present invention provides a method for preparing a glycoconjugate, comprising:

Selectively oxidize the primary hydroxyl groups of bacterial capsular polysaccharides to aldehyde groups with nitroxyl compounds and iodine oxidants to obtain bacterial capsular polysaccharides containing aldehyde groups;

Optionally, reacting the bacterial capsular polysaccharide containing an aldehyde group with a spacer to obtain a bacterial capsular polysaccharide derivative containing an aldehyde group;

Prepare the glycoconjugate by reacting the aldehyde-containing bacterial capsular polysaccharide or the aldehyde-containing bacterial capsular polysaccharide derivative with the amino group or carboxyl group of the carrier protein, preferably, the aldehyde-containing bacterial capsular polysaccharide derivative and the carrier Proteins were reacted in the presence of EDAC to prepare glycoconjugates.

Preferably, the bacterial capsular polysaccharide is selected from Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C , 19A, 19F, 20, 22F, 23F, 33F type capsular polysaccharide, further preferably, the bacterial capsular polysaccharide is Streptococcus pneumoniae type 12F capsular polysaccharide.

Preferably, the activation site of the bacterial capsular polysaccharide is the primary hydroxyl group of acetylaminogalactosyl (α-D-GalpNAc), the primary hydroxyl group of galactopyranosyl (α-D-Galp), or the primary hydroxyl group of pyranosyl Glucopyranosyl Primary Hydroxyl (α-D-Glcp).

Preferably, the iodine oxidizing agent is iodobenzenediacetic acid (PIA).

Preferably, the nitroxyl compound is 2,2,6,6-tetramethyl-1-piperidinyloxy radical or contains 2,2,6,6-tetramethyl-1-piperidinyloxy Compounds with a free radical structure.

Preferably, the compound containing 2,2,6,6-tetramethyl-1-piperidinyloxy radical structure includes but not limited to: 2,2,6,6-tetramethylpiperidinyl oxide , 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinyloxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-iso Thiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO radical, 4-hydroxy-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl, 4-cyano-TEMPO, 4-methoxy-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO or 4-amino-TEMPO.

Preferably, the spacer contains the general formula Y ₁ -LY ₂ , wherein Y ₁ contains the first primary amino group that can react with the carbonyl (or carboxyl) in the polysaccharide; Y ₂ contains an ester that can react with the linker and L is the linking moiety in the other linker. Typical L groups are straight chain alkyl groups having 1-10 carbon atoms (eg C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ ) , especially -(CH ₂ ) ₄ -. Homobifunctional linkers of the general formula YLY are particularly suitable as spacers, wherein the two Y groups are identical and are both capable of reacting with carbonyl (or carboxyl) and ester groups; and wherein L is the linkage in the spacer part. Typically the Y group is a -NHNH2 group. L generally has the general formula -L'- ^L2 -L'-, where L' is carbonyl. Typically the ^L2 group is a straight chain alkyl having 1-10 carbon atoms (e.g. _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 ) , especially -(CH ₂ ) ₄ -.

Preferably, the spacer is selected from ADH (adipate dihydrazide) or CDH (carboxydihydrazine).

In a specific embodiment of the present invention, the spacer is ADH.

Preferably, the molar ratio of the nitroxyl compound to the capsular polysaccharide=(0.03～0.1):1 (for example, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1 , 0.09:1, 0.1:1).

Further preferably, the molar ratio of the nitroxyl compound to the capsular polysaccharide=(0.03-0.08):1.

Preferably, the molar ratio of the iodine oxidant to the capsular polysaccharide=(0.5-5):1 (for example, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3: 1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8: 1, 4.9:1, 5:1).

Further preferably, the molar ratio of the iodine oxidizing agent to the capsular polysaccharide=(0.8-3):1.

Preferably, the molar ratio of activated ADH to capsular polysaccharide is 3 to 96 (such as 3, 5, 7, 9, 10, 15, 20, 24, 25, 30, 35, 40, 45, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96).

Further preferably, the molar ratio of ADH to activated capsular polysaccharide is 24-48.

Preferably, unreacted aldehyde groups are reduced to hydroxyl groups in a blocking reaction using sodium borohydride after conjugation to the carrier protein either directly or via a spacer, thereby allowing the sugar surface to Bit modification is minimal.

Preferably, after the unreacted aldehyde group has reacted with the protein, adjust the pH to 7.0 with 0.1M sodium hydroxide to block the reaction.

Preferably, the nitroxyl compound has the ability to selectively oxidize primary hydroxyl groups to generate aldehyde groups in the presence of the oxidizing agent.

Preferably, the oxidizing agent is an oxidizing agent with hypervalent iodine capable of selectively oxidizing primary hydroxyl groups to generate aldehyde groups in the presence of nitroxyl compounds.

Preferably, the carrier protein contains one or more amino/carboxyl groups. The carrier protein can be a related protein antigen from the target pathogen that enhances the specific immune response to the pathogen, or a general immunogenic protein that mainly acts as an adjuvant or general immune response stimulator.

Further preferably, the carrier protein is selected from the group consisting of CRM197, tetanus toxoid, outer membrane protein from Gram-negative bacteria, surface lipoprotein (HiD) of Haemophilus influenzae, HiD protein gene from Haemophilus influenzae and Fusion protein of Haemophilus influenzae Hin47 protein gene in a 1:1 manner, pertussis toxin, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein or respiratory syncytial virus F and G proteins or their active parts .

In a specific embodiment of the present invention, the carrier protein is CRM197 or TT.

Preferably, the Streptococcus pneumoniae 12F capsular polysaccharide can be natural or synthetic.

Preferably, the molar ratio of TEMPO to capsular polysaccharide=(0.03～0.1):1 (for example, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09 :1, 0.1:1).

Further preferably, the molar ratio of TEMPO to capsular polysaccharide=(0.03-0.085):1.

Preferably, the molar ratio of PIA to capsular polysaccharide=(0.05～5):1 (for example, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4: 1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9: 1, 5:1).

Further preferably, the molar ratio of PIA to capsular polysaccharide=(0.8-3):1.

Preferably, the oxidation of primary hydroxyl to aldehyde is carried out in an aqueous solvent.

Preferably, the nitroxyl compound is used in a catalytic amount≤0.1 molar equivalent, and the desired degree of oxidation of the capsular polysaccharide can be achieved by changing the amount of the oxidizing agent used.

Preferably, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3 (for example, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3).

Further preferably, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8 to 1.7.

In a specific embodiment of the present invention, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 1.0 to 1.5.

In a specific embodiment of the present invention, the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8 to 1.2.

Preferably, for every 10 to 30 sugar repeating units of the bacterial capsular polysaccharide, there is at least one covalent bond between the carrier protein and the bacterial capsular polysaccharide.

Preferably, the oxidation (activation) time is 3 to 24 hours.

In a specific embodiment of the present invention, the oxidation time is 12 to 24 hours.

In a specific embodiment of the present invention, the preparation method of the glycoconjugate comprises:

1) hydrolyzing the 12F type Streptococcus pneumoniae capsular polysaccharide to a molecular weight of 50-500kDa, using TEMPO and PIA as activators to oxidize the primary hydroxyl on the sugar ring to an aldehyde group, and obtain the activated 12F type Streptococcus pneumoniae capsular polysaccharide; Preferably, the degree of activation of the polysaccharide is from 5 to 20, more preferably, the degree of oxidation of the activated Streptococcus pneumoniae type 12F capsular polysaccharide is from 5 to 15, more preferably, the degree of activation is from 6 to 12. Preferably, the activation reaction time is 2 to 24 hours. Further preferably, the activation reaction time is 16 to 20 hours.

2) The 12F Streptococcus pneumoniae capsular polysaccharide activated in step 1) was reacted with TT or CRM197 to prepare a glycoconjugate. In this, unreacted aldehyde groups are reduced to primary hydroxyl groups in a blocking reaction using sodium borohydride after conjugation to the carrier protein.

3) Purifying the glycoconjugate obtained in step 2).

Preferably, in the step 1), the 12F Streptococcus pneumoniae capsular polysaccharide is hydrolyzed to a molecular weight of 50-500 kDa.

Preferably, the step 1) further includes the step of purifying the activated Streptococcus pneumoniae type 12F capsular polysaccharide.

Preferably, the temperature of the blocking reaction in step 2) is 20 to 35°C.

Preferably, the blocking reaction time in the step 2) is 2 to 6 hours.

Preferably, the method is applicable to any primary hydroxyl group-containing capsular polysaccharide of Streptococcus pneumoniae serotype.

In another specific embodiment of the present invention, the preparation method of the glycoconjugate comprises:

1) Hydrolyzing the 12F Streptococcus pneumoniae capsular polysaccharide to a molecular weight of 50-500kDa, using TEMPO and PIA as activators to oxidize the primary hydroxyl on the sugar ring to an aldehyde group to obtain an activated polysaccharide; preferably, the polysaccharide The degree of activation is 5 to 20. More preferably, the degree of oxidation of the activated 12F capsular polysaccharide is 5 to 15. Even more preferably, the degree of activation is 6 to 12. Preferably, the activation reaction time is 2 to 24 hours. Further preferably, the activation reaction time is 16 to 20 hours.

2) The 12F-type Streptococcus pneumoniae capsular polysaccharide activated in step 1) was reacted with ADH to prepare 12F-derived polysaccharide. Wherein, the unreacted ADH was ultrafiltered 30 times with 10 mM phosphate buffer solution after the reaction, and then ultrafiltered with water for 10 times, so as to be completely removed.

3) Preparation of glycoconjugates: reacting the derivatized polysaccharide obtained in step 2) with carrier protein TT or CRM197 to obtain a conjugate, and purifying the obtained conjugate with AKTA to obtain a stock solution of the conjugate;

Preferably, in the step 1), the 12F Streptococcus pneumoniae capsular polysaccharide is hydrolyzed to a molecular weight of 50-500 kDa.

Preferably, the step 1) further includes the step of purifying the activated 12F type Streptococcus pneumoniae capsular polysaccharide.

Preferably, the reaction temperature in step 2) is 20 to 40°C.

Preferably, the blocking reaction time in the step 2) is 2 to 6 hours.

The third aspect of the present invention provides a glycoconjugate prepared by the above-mentioned preparation method.

The fourth aspect of the present invention provides an immunogenic composition, comprising the above-mentioned glycoconjugate or the glycoconjugate prepared by the above-mentioned preparation method, and a pharmaceutically acceptable excipient, carrier and/or diluent .

Preferably, the composition includes a Streptococcus pneumoniae 12F type capsular polysaccharide conjugate, and the Streptococcus pneumoniae 12F type capsular polysaccharide conjugate uses a nitroxyl compound and an iodine oxidizing agent to convert the 12F type bacterial capsular polysaccharide Primary hydroxyl group is selectively oxidized to aldehyde group to obtain 12F type bacterial capsular polysaccharide containing aldehyde group;

Optionally, reacting the 12F type bacterial capsular polysaccharide containing aldehyde group with the spacer to obtain the 12F type bacterial capsular polysaccharide derivative containing aldehyde group;

It is prepared by reacting the 12F type bacterial capsular polysaccharide containing aldehyde group or the 12F type bacterial capsular polysaccharide derivative containing aldehyde group with the amino group or carboxyl group of the carrier protein.

Preferably, the immunogenic composition further comprises glycoconjugates of other bacterial capsular polysaccharides selected from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, and 6A , 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F capsular polysaccharide.

Preferably, the dosage form of the immunogenic composition is selected from the group consisting of tablets, capsules, pills, injections, inhalants, buccal tablets, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, Suspoemulsion, cream, jelly, spray, etc.

Preferably, the administration mode of the immunogenic composition is selected from: oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal cavity administration, transdermal administration, subconjunctival administration, intraocular administration , orbital administration, retrobulbar administration, retinal administration, choroidal administration, intrathecal injection, etc.

Preferably, the immunogenic composition further includes an adjuvant. More preferably, the adjuvant is an aluminum adjuvant. Most preferably, the aluminum-based adjuvant is selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide.

Preferably, the immunogenic composition further comprises physiological saline and succinic acid.

The fifth aspect of the present invention provides the above-mentioned glycoconjugate, the glycoconjugate prepared by the above-mentioned preparation method or the above-mentioned immunogenic composition in the prevention and/or treatment of individual Streptococcus pneumoniae infection, and pneumoniae Drugs or vaccines for cocci-related diseases. The glycoconjugates and immunogenic compositions provided by the invention have high immunogenicity and can induce therapeutic immune responses in individuals.

Preferably, the disease associated with Streptococcus pneumoniae is selected from pneumonia, meningitis, cellulitis, osteomyelitis, endocarditis, septic shock, febrile bacteremia, middle ear infection, sinusitis, recurrent type bronchitis and other severe invasive diseases.

The sixth aspect of the present invention provides a drug for preventing and/or treating individual Streptococcus pneumoniae infection and Streptococcus pneumoniae-related diseases, the drug comprising the glycoconjugate or immunogenicity of the present invention combination.

The seventh aspect of the present invention provides a vaccine for preventing and/or treating individual Streptococcus pneumoniae infection and Streptococcus pneumoniae-related diseases, said vaccine comprising the glycoconjugate or immunogenicity of the present invention combination.

Preferably, the vaccine is a liquid injection.

Preferably, the injection also contains physiological saline, succinic acid, aluminum phosphate adjuvant and the like.

Preferably, the vaccine comprises at least serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C of Streptococcus pneumoniae , 19A, 19F, 20, 22F, 23F, 33F capsular polysaccharides.

Preferably, each dose of the vaccine contains 1 to 5 μg of polysaccharide.

The eighth aspect of the present invention provides a method for preventing and/or treating individual Streptococcus pneumoniae infection and Streptococcus pneumoniae-related diseases, the method comprising administering to the individual an effective dose of the glycoconjugated compounds or immunogenic compositions.

The "glycoconjugate" in the present invention refers to a sugar covalently conjugated to a carrier protein. Wherein, the glycoconjugate may contain a certain amount of free sugar.

The "activation degree" mentioned in the present invention refers to the molar ratio of sugar repeating units per mole of aldehyde.

The "derivatization rate" in the present invention refers to the ratio of ADH concentration (μg/ml) to polysaccharide concentration (mg/ml).

The "binding ratio" in the present invention refers to the ratio of the polysaccharide concentration (mg/ml) to the protein concentration (mg/ml) in the conjugate.

"Pharmaceutically acceptable" in the present invention means neither significantly stimulating the individual nor inhibiting the biological activity and characteristics of the administered active substance.

The "prevention" in the present invention refers to all actions of suppressing symptoms or delaying the tension of specific symptoms by administering the products described in the present invention.

"Treatment" as used herein refers to therapeutic intervention to ameliorate the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.

The "individual" in the present invention includes mammals and humans.

The "effective dose" in the present invention refers to the amount or dose of the composition of the present invention that provides the desired treatment or prevention after being administered to an individual or an organ in single or multiple doses.

The "and/or" in the present invention includes alternatively listed items and any number of item combinations.

The "comprising" in the present invention is an open-ended description, containing the described specified components or steps, and other specified components or steps that do not substantially affect.

The above only summarizes some aspects of the present invention, and it is not and should not be considered as limiting the present invention in any respect.

All patents and publications mentioned in this specification are hereby incorporated by reference in their entirety. Those skilled in the art would recognize that certain changes can be made in the present invention without departing from the spirit or scope of the invention.

The following examples further illustrate the present invention and should not be considered as limiting the scope of the present invention or the specific methods described in the present invention.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail in conjunction with the accompanying drawings, wherein:

Figure 1: Figures A and B represent the structural formula and expression of the capsular polysaccharide of Streptococcus pneumoniae serotype 12F, respectively, where the position of the ellipse in Figure A indicates the oxidation site of the capsular polysaccharide oxidized by TEMPO-PIA, respectively β -D-GalpNAc, α-D-Galp, α-D-Glcp, primary hydroxyl; the position of the rectangular box indicates the site of periodate-mediated oxidation, specifically α-D-Galp, α-D-Glcp , o-dihydroxyl;

Figure 2: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 2;

Figure 3: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 6B;

Figure 4: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 8;

Figure 5: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 9N;

Figure 6: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 10A;

Figure 7: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 11A;

Figure 8: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 15B;

Figure 9: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 17A;

Figure 10: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 17F;

Figure 11: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 20;

Figure 12: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 22F;

Figure 13: Structural formula and expression of capsular polysaccharide of Streptococcus pneumoniae serotype 33F;

Figure 14: ELISA method to detect the immunogenicity results of 12F polysaccharide conjugates;

Figure 15: The immunogenicity results of 12F polysaccharide conjugates detected by MOPA method;

Figure 16: NMR structures of 12F-related products prepared by different processes, where NAME is the name and number of 12F-related products, EXPNO is the experiment number, PROCNO is the treatment number, INSTRUM is the name of the cabinet, PROBHD is the probe model, and PULPROG is the pulse sequence. TD is the number of sampling points, SOLVENT is the solvent, NS is the number of scans, DS is the number of empty scans, SWH is the spectral width, FIDRES is the resolution of the free induction attenuation signal, AQ is the sampling time, RG is the receiver gain, and DW is the sampling interval , DE is the time interval when the transmitter is turned off and the receiver is turned on, D1 is the cycle delay, TD0 represents how many times it is saved, SFO1 is the fundamental frequency + offset of the observation channel, NUC1 is the core of the observation channel, P1 is the pulse width, PLW1 is the power, SI is the number of points after Fourier transform, SF is the fundamental frequency, WDW is the window function, EM refers to the exponential window function, SSB is the parameter of the sinusoidal ringing window function, LB is the linear broadening factor, and GB is The parameters of the Gaussian window function, PC is the peak detection sensitivity;

Figure 16A: NMR structure of 12F activated polysaccharide (F12F-A-20210309) prepared by TEMPO-PIA process;

Figure 16B: NMR structure of 12F-derived polysaccharide (F12F-AA-20210312) prepared by TEMPO-PIA process;

Figure 16C: NMR structure of 12F activated polysaccharide (F12F-A-20210220) prepared by TEMPO-NCS process;

Figure 16D: NMR structure of 12F-derived polysaccharide (F12F-AA-20210122) prepared by TEMPO-NCS process;

Figure 16E: NMR structure of 12F-derived polysaccharide (F12F-AA-20210219-2) prepared by TEMPO-NCS process;

Figure 16F: NMR structure of 12F-derived polysaccharide (F12F-AA-20210222-2) prepared by TEMPO-NCS process;

Figure 17: The particle size of the 12F glycoconjugate prepared in Example 1;

Figure 18: The particle size of the 12F glycoconjugate prepared in Example 2;

Figure 19: The particle size of the 12F glycoconjugate prepared in Example 4;

Figure 20: ADH methylene characteristic peak, NMR shift;

Figure 21: Comparison of TEMPO-PIA process-related spectra;

Figure 22: Comparison of TEMPO-NCS process-related spectra.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Preparation of serotype 12F-CRM197 glycoconjugate using TEMPO-PIA (1)

In order to improve the stability of the 12F polysaccharide vaccine process and the immunogenicity of the conjugate, the inventors used 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) and its co-oxidant iodobenzene di Ethyl ester (iodobenzenediacetic acid or PIA) oxidizes polysaccharide primary hydroxyl to aldehyde, and then reacts with amino group of carrier protein to prepare 12F-CRM197 conjugate.

Among them, the main oxidant is PIA, and the by-product is iodobenzene; TEMPO is the catalytic amount, and the by-product is 1-hydroxy-2,2,6,6-tetramethylpiperidine. Analysis shows that the oxidation (activation or reaction) site of this method is different from the oxidation site of sodium periodate. The oxidation site of TEMPO-PIA is the primary hydroxyl group on α-D-GalpNAc, α-D-Galp, and α-D-Glcp (see Figure 1), and the oxidation site of sodium periodate is mainly α-D-Galp On the adjacent dihydroxyl (see Figure 1).

Among the present invention, the possible reaction mechanism of iodobenzenediethyl ester (iodobenzenediacetic acid) oxidation primary hydroxyl is as follows:

The method for preparing glycoconjugates of serotype 12F-CRM197 is as follows:

1) Add 12F capsular polysaccharide to acetic acid solution, control pH = 2, and heat for 2 hours to obtain 12F hydrolyzed polysaccharide; TSK molecular weight measurement, molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 250 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 2 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (2.2mg, 0.06eq) and PIA (220mg, 3eq), protected from light, reacted at room temperature for 20 hours.

Ultrafiltration, 30KD membrane bag ultrafiltration 50 times, 205 mg of activated polysaccharide was obtained, the yield was 82%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Combine 12F activated polysaccharide with CRM197 carrier protein

Freeze-dry at -40°C for 72 hours to obtain the activated polysaccharide solid.

Dissolve 200mg of 12F activated polysaccharide in water to a final concentration of 10mg/ml, add an equal mass of CRM197 carrier protein, mix well, add sodium cyanoborohydride (23mg, 2eq), protect from light, and react at 37°C for 72 hours. After the reaction was completed, sodium borohydride (14mg, 2eq) was added to block the reaction for 1-3h.

Purification: 100KD membrane bag, 50 times of ultrafiltration of 10mM phosphate buffer solution, 20 times of ultrafiltration of 0.9% sodium chloride solution, to obtain the stock solution of the conjugate.

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The protein content was measured by the Folin phenol method, the polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by the SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The specific test data of the experiment can be seen in Table 1.

At the same time, the particle size of the final product was detected by dynamic light scattering (Dynamic lightscattering, DLS), and the results are shown in Figure 17.

Example 2 Preparation of serotype 12F-CRM197 glycoconjugates using TEMPO-PIA and ADH

The method for preparing glycoconjugates of serotype 12F-CRM197 is as follows:

1) Hydrolyze the 12F capsular polysaccharide under acidic heating conditions to obtain the 12F hydrolyzed polysaccharide, and measure the molecular weight by TSK. The molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 175 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 4 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (1mg, 0.04eq) and PIA (43.8mg, 0.85eq) were reacted at room temperature for 20 hours in the dark.

Ultrafiltration, 30KD membrane bag, pure water ultrafiltration 30 times, 127mg of activated polysaccharide was obtained, the yield was 72.6%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Preparation of 12F-derived polysaccharides

Dissolve 250 mg of 12F activated polysaccharide in water to a final concentration of 8 mg/ml. Phosphate buffer at pH = 6.5 was added to a final concentration of 10 mM. ADH solid (1910mg, 48eq) was added and stirred to dissolve. Sodium cyanoborohydride (28.7mg, 2eq) was added, protected from light, and reacted at a constant temperature of 37°C for 8-16h. After the reaction was completed, sodium borohydride (17.3mg, 2eq) was added to block the reaction for 1-3h.

Ultrafiltration: 30KD membrane bag, 50 times of ultrafiltration with 10mM phosphate buffer solution, 20 times of ultrafiltration with pure water, and concentration to obtain 203mg of derivatized polysaccharide with a yield of 81.3%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, NMR was used to detect capsular polysaccharide structure, and pharmacopoeia method was used to measure ADH content (Chinese Pharmacopoeia General Rules 3118 Determination of adipic hydrazide content). The ratio of ADH content (unit μg/ml) to polysaccharide content (unit mg/ml) is the ADH derivation rate.

4) Combining 12F-derived polysaccharides with CRM197 carrier protein

Dissolve 200mg of 12F-derived polysaccharide in water to a final concentration of 4mg/ml, control the temperature in an ice bath at 2-8°C, add an equal mass of CRM197 carrier protein, mix well, add EDAC to a final concentration of 20mg/ml, adjust with hydrochloric acid pH = 5.3, react for 3 hours, after the reaction, adjust pH = 6.6-7.5 with sodium hydroxide, and block the reaction for 1 hour. After filtering with a 0.45 μm filter, it was purified by AKTA to obtain the stock solution of the conjugate.

The protein content was measured by the Folin phenol method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The specific test data of the experiment can be seen in Table 1.

At the same time, the particle size of the final product was detected by dynamic light scattering (Dynamic lightscattering, DLS), and the results are shown in Figure 18.

Example 3 Preparation of serotype 12F-TT glycoconjugates using TEMPO-PIA and ADH

The method for preparing serotype 12F-TT conjugates is as follows

The method of this example is similar to Example 2, but the carrier protein is tetanus toxoid.

The method for preparing serotype 12F-TT conjugates is as follows:

1) Hydrolyze the 12F capsular polysaccharide under acidic heating conditions to obtain the 12F hydrolyzed polysaccharide, and measure the molecular weight by TSK. The molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 350 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 2 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (2mg, 0.04eq) and PIA (87.6mg, 0.85eq) were reacted at room temperature for 20 hours in the dark.

Ultrafiltration, 30KD membrane bag, pure water ultrafiltration 30 times, 254 mg of activated polysaccharide was obtained, and the yield was 72.6%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Preparation of 12F-derived polysaccharides

Dissolve 250 mg of 12F activated polysaccharide in water to a final concentration of 5 mg/ml. Phosphate buffer at pH = 6.5 was added to a final concentration of 10 mM. ADH solid (1910mg, 48eq) was added and stirred to dissolve. Sodium cyanoborohydride (28.7mg, 2eq) was added, protected from light, and reacted at a constant temperature of 37°C for 8-16h. After the reaction was completed, sodium borohydride (17.3mg, 2eq) was added to block the reaction for 1-3h.

Ultrafiltration: 30KD membrane bag, 50 times of ultrafiltration with 10mM phosphate buffer solution, 20 times of ultrafiltration with pure water, concentrated to obtain 209 mg of derivatized polysaccharide, with a yield of 83.6%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, NMR was used to detect capsular polysaccharide structure, and TNBS method was used to measure ADH content (Chinese Pharmacopoeia General Rules 3118 Adipic Dihydrazide Content Determination Method). The ratio of ADH content (unit μg/ml) to polysaccharide content (unit mg/ml) is the ADH derivation rate.

4) Combining 12F-derived polysaccharides with TT carrier protein

Dissolve 200mg of 12F-derived polysaccharide in water to a final concentration of 4mg/ml, control the temperature in an ice bath at 2-8°C, add 1.1 times the mass of TT carrier protein, mix well, add EDAC to a final concentration of 20mg/ml, hydrochloric acid Adjust the pH=5.3±0.2, react for 1-3 hours, after the reaction, adjust the pH=6.6-7.5 with sodium hydroxide, and block the reaction for 1 hour. After filtering with a 0.22 μm filter, AKTA was purified to obtain the stock solution of the conjugate.

The protein content was measured by the Folin phenol method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The specific test data of the experiment can be seen in Table 1.

Example 4 Preparation of serotype 12F-CRM197 glycoconjugates using TEMPO-NCS and ADH

Preparation of serotype 12F-CRM197 glycoconjugates.

1) Hydrolyze the 12F capsular polysaccharide under acidic heating conditions to obtain the 12F hydrolyzed polysaccharide, and measure the molecular weight with TSK. The molecular weight should be controlled at 50-500kDa;

2) TEMPO-NCS activated polysaccharide, the specific operation is: under ice bath, dissolve 300mg of 12F hydrolyzed polysaccharide in water to a final concentration of 2mg/ml, add pH 8.6 sodium carbonate-sodium bicarbonate buffer to a final concentration of 10mM , add TEMPO (3.6mg, 0.085eq) and NCS (73mg, 2eq), and react at 2-8°C for 3 hours in the dark.

Ultrafiltration, 30KD membrane bag pure water ultrafiltration 50 times, concentrated to obtain 235mg of activated polysaccharide, yield 78.3%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Preparation of 12F-derived polysaccharides

Dissolve 250 mg of 12F activated polysaccharide in water to a final concentration of 5 mg/ml. Phosphate buffer at pH = 6.5 was added to a final concentration of 10 mM. ADH solid (1910mg, 48eq) was added and stirred to dissolve. Sodium cyanoborohydride (28.7mg, 2eq) was added, protected from light, and reacted at a constant temperature of 37°C for 8-16h. After the reaction was completed, sodium borohydride (17.3mg, 2eq) was added to block the reaction for 1-3h.

Ultrafiltration: 30KD membrane bag, 50 times of ultrafiltration with 10mM phosphate buffer solution, 20 times of ultrafiltration with pure water, and concentration to obtain 220mg of derivatized polysaccharide with a yield of 88.0%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, NMR was used to detect capsular polysaccharide structure, and pharmacopoeia method was used to measure ADH content (Chinese Pharmacopoeia General Rules 3118 Adipic Dihydrazide Content Determination Method). The ratio of ADH content (unit μg/ml) to polysaccharide content (unit mg/ml) is the ADH derivation rate.

Dissolve 200mg of 12F-derived polysaccharide in water to a final concentration of 4mg/ml, control the temperature in an ice bath at 2-8°C, add an equal mass of CRM197 carrier protein, mix well, add EDAC to a final concentration of 20mg/ml, adjust with hydrochloric acid pH = 5.4, react for 3 hours, after the reaction, adjust pH = 6.6-7.5 with sodium hydroxide, and block the reaction for 1 hour. After filtering with a 0.45 μm filter, it was purified by AKTA to obtain the stock solution of the conjugate.

The protein content was measured by the Folin method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The specific test data of the experiment can be seen in Table 1.

At the same time, the particle size of the final product was detected by dynamic light scattering (Dynamic lightscattering, DLS), and the results are shown in Figure 19.

Example 5 Preparation of serotype 12F-TT glycoconjugates using TEMPO-NCS and ADH

Preparation of serotype 12F-TT glycoconjugates.

1) Hydrolyze the 12F capsular polysaccharide under acidic heating conditions to obtain 12F hydrolyzed polysaccharide, measure the molecular weight with TSK, and the molecular weight should be controlled at 50-200KD;

2) TEMPO-NCS activated polysaccharide, the specific operation is: under ice bath, dissolve 300mg of 12F hydrolyzed polysaccharide in water to a final concentration of 2mg/ml, add pH 8.6 sodium carbonate-sodium bicarbonate buffer to a final concentration of 10mM , add TEMPO (3.6mg, 0.085eq) and NCS (73mg, 3eq), and react at room temperature for 3 hours in the dark.

Ultrafiltration, 30KD membrane bag pure water ultrafiltration 50 times, concentrated to obtain 255mg of activated polysaccharide, yield 85.0%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Preparation of 12F-derived polysaccharides

Dissolve 250 mg of 12F activated polysaccharide in water to a final concentration of 5 mg/ml. Phosphate buffer at pH = 6.5 was added to a final concentration of 10 mM. ADH solid (1910mg, 48eq) was added and stirred to dissolve. Sodium cyanoborohydride (28.7mg, 2eq) was added, protected from light, and reacted at a constant temperature of 37°C for 8-16h. After the reaction was completed, sodium borohydride (17.3mg, 2eq) was added to block the reaction for 1-3h.

Ultrafiltration: 30KD membrane bag, 50 times of ultrafiltration with 10mM phosphate buffer solution, 20 times of ultrafiltration with pure water, and concentration to obtain 217 mg of derivatized polysaccharide, with a yield of 86.8%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, NMR was used to detect capsular polysaccharide structure, and pharmacopoeia method was used to measure ADH content (Chinese Pharmacopoeia General Rules 3118 Adipic Dihydrazide Content Determination Method). The ratio of ADH content (unit μg/ml) to polysaccharide content (unit mg/ml) is the ADH derivation rate.

Dissolve 200mg of 12F-derived polysaccharide in water to a final concentration of 4mg/ml, control the temperature in an ice bath at 2-8°C, add 1.2 times the mass of TT carrier protein, mix well, add EDAC to a final concentration of 20mg/ml, hydrochloric acid Adjust the pH to 5.4 and react for 3 hours. After the reaction, adjust the pH to 6.6-7.5 with sodium hydroxide and block the reaction for 1 hour. After filtering with a 0.45 μm filter, purify with AKTA to obtain the stock solution of the conjugate.

The protein content was measured by the Folin method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The specific test data of the experiment can be seen in Table 1.

Example 6 Preparation of serotype 12F-CRM197 glycoconjugate using TEMPO-PIA (2)

The method for preparing glycoconjugates of serotype 12F-CRM197 is as follows:

1) Add 12F capsular polysaccharide to acetic acid solution, control pH = 2, and heat for 2 hours to obtain 12F hydrolyzed polysaccharide; TSK molecular weight measurement, molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 250 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 2 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (3.0mg, 0.08eq) and PIA (220mg, 3eq) were reacted at room temperature in the dark for 16 hours.

Ultrafiltration, 30KD membrane bag ultrafiltration 50 times, to obtain 220 mg of activated polysaccharide, yield 88.0%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Combine 12F activated polysaccharide with CRM197 carrier protein

Mix 200 mg of 12F activated polysaccharide with 1.2 times the mass of CRM197 protein, freeze in a -80°C refrigerator for 8 hours, and then vacuum freeze-dry it at -40°C for 72 hours to obtain a freeze-dried powder.

Dissolve the lyophilized powder in water until the sugar concentration is 10mg/ml, add sodium cyanoborohydride (23mg, 2eq), protect from light, and react at 37°C for 72 hours. After the reaction was completed, sodium borohydride (14mg, 2eq) was added to block the reaction for 1-3h.

Purification: 100KD membrane bag, 50 times of ultrafiltration of 10mM phosphate buffer solution, 20 times of ultrafiltration of 0.9% sodium chloride solution, to obtain the stock solution of the conjugate.

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The protein content was measured by the Folin method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The specific test data of the experiment can be seen in Table 1.

Example 7 Preparation of serotype 12F-CRM197 glycoconjugate using TEMPO-PIA (3)

The method for preparing glycoconjugates of serotype 12F-CRM197 is as follows:

1) Add 12F capsular polysaccharide to acetic acid solution, control pH = 2, and heat for 2 hours to obtain 12F hydrolyzed polysaccharide; TSK molecular weight measurement, molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 250 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 2 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (2.2mg, 0.06eq) and PIA (220mg, 3eq), protected from light, reacted at 2-8°C for 3 hours.

Ultrafiltration, 30KD membrane bag ultrafiltration 50 times, to obtain 213mg of activated polysaccharide, yield 85%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Combine 12F activated polysaccharide with CRM197 carrier protein

Mix the 12F activated polysaccharide with 25 times the mass of sucrose, and place it at -40°C for 72 hours in a vacuum freeze-dry to obtain a solid activated polysaccharide.

Dissolve 200mg of 12F activated polysaccharide in water to a final concentration of 10mg/ml, add an equal mass of CRM197 carrier protein, mix well, add sodium cyanoborohydride (23mg, 2eq), protect from light, and react at 37°C for 72 hours. After the reaction was completed, sodium borohydride (14mg, 2eq) was added to block the reaction for 1-3h.

Purification: 100KD membrane bag, 10mM phosphate buffer solution ultrafiltration 50 times, 0.9% sodium chloride solution ultrafiltration 20 times, to obtain the stock solution of the conjugate.

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The protein content was measured by the Folin method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The specific test data of the experiment can be seen in Table 1.

Example 8 Preparation of serotype 12F-CRM197 glycoconjugate using TEMPO-PIA (four)

The method for preparing glycoconjugates of serotype 12F-CRM197 is as follows:

1) Add 12F capsular polysaccharide to acetic acid solution, control pH = 2, and heat for 2 hours to obtain 12F hydrolyzed polysaccharide; TSK molecular weight measurement, molecular weight should be controlled at 50kDa to 500kDa;

2) TEMPO-PIA activates polysaccharides, the specific operation is: under ice bath, dissolve 250 mg of 12F hydrolyzed polysaccharides in water to a final concentration of 2 mg/ml, add pH=6.5 phosphate buffer to a final concentration of 10 mM, add TEMPO (1.5mg, 0.04eq) and PIA (75mg, 1eq) were reacted at room temperature for 3 hours in the dark.

Ultrafiltration, 30kd membrane bag ultrafiltration 50 times, to obtain 200 mg of activated polysaccharide, yield 80.0%. Anthrone method was used to measure sugar content, MBTH method was used to measure aldehyde group content, TSK was used to measure molecular weight, and NMR was used to detect capsular polysaccharide structure. The ratio of sugar content to aldehyde content is the degree of activation (DO).

3) Combine 12F activated polysaccharide with CRM197 carrier protein

The 12F activated polysaccharide was vacuum freeze-dried at -40°C for 72 hours to obtain a solid activated polysaccharide.

Dissolve 200mg of 12F activated polysaccharide in water to a final concentration of 12mg/ml, add 1.2 times the mass of CRM197 carrier protein, mix well, add sodium cyanoborohydride (23mg, 2eq), protect from light, and react at 37°C for 72 hours . After the reaction was completed, sodium borohydride (14mg, 2eq) was added to block the reaction for 1-3h.

Purification: 100KD membrane bag, 50 times of ultrafiltration of 10mM phosphate buffer solution, 20 times of ultrafiltration of 0.9% sodium chloride solution, to obtain the stock solution of the conjugate.

The stock solution of the above conjugate was sterile filtered with a 0.2 μm filter, and this stock solution material was called a monovalent conjugate. All monovalent conjugates of primary hydroxyl-containing serotypes can be generated in a similar manner.

The protein content was measured by the Folin method, the capsular polysaccharide content was measured by the anthrone method, the free polysaccharide content was measured by the DOC precipitation method, the free protein content was measured by SDS-PAGE method, and the molecular weight was measured by CL-4B (K _D ≤0.3).

The specific test data of the experiment can be seen in Table 1.

Example 9 Preparation of glycoconjugates of various serotypes containing primary hydroxyl groups

Methods of preparing glycoconjugates of various serotypes containing primary hydroxyl groups:

The experimental operation is the same as in Examples 1-8.

Serotypes containing primary hydroxyl groups include, but are not limited to, those described herein.

This method can be applied to other serotypes. The structures and expressions of other blood types are shown in Figure 2-13, and the primary hydroxyl groups are all selectively oxidized:

Type 2 reaction site D-Glcp, type 3 reaction site β-D-Glcp, type 4 reaction site β-D-ManpNAc, etc., type 5 reaction site D-Glcp, type 6A reaction site D-Galp, D-Glcp, 6B type reaction site D-Glcp, D-Glcp, 7F type reaction site D-Galp, etc., 8 type reaction site β-D-Glcp, etc., 9N type reaction site α-D-Glup, etc. , 9V type reaction site α-D-Galp etc., 10A type reaction site β-D-Galp etc., 11A type reaction site β-D-Galp etc., 14 type reaction site D-Glcp etc., 15B type reaction site Site α-D-Galp etc., Type 17A Reaction Site β-D-Glcp etc., Type 17F Reaction Site β-D-Glcp etc., Type 18C Reaction Site D-Glcp etc., Type 19A Reaction Site β- D-ManpNAc, etc., 19F-type reactive sites β-D-ManpNAc, etc., 20-type reactive sites α-D-GlcpNAc, etc., 22F-type reactive sites α-D-Glcp, 23F-type reactive sites D-Glcp, L -Rhp, 33F-type reactive site β-D-Galp, etc.

Table 1: Data Summary Table of Examples 1-8

Example 10 Preparation of pneumococcal 24-valent polysaccharide conjugate vaccine

Formulation of polysaccharide-CRM197 conjugates as vaccines

24 pneumococcal conjugate stocks (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20 , 22F, 23F, 33F) were added in 0.9% normal saline according to a certain proportion, fully mixed, and an appropriate amount of 50mM succinic acid solution and aluminum phosphate adjuvant were added to prepare a pneumococcal 24-valent polysaccharide conjugate vaccine.

Among them, the content of various types of polysaccharides is 2.2 μg/ml (6B 4.4 μg/ml), the content of aluminum ions is not higher than 0.2 mg/ml, and it is packaged in 0.5 ml/cartridge.

Example 11 Immunogenicity Research

For this study, ordinary rabbits were recruited, about 2.5kg each, half male and half male. The experiment consisted of 10 groups: 8 experimental groups, 1 positive control vaccine group (Prevenar13), and 1 placebo group. Give rabbits a dose volume of 0.5ml, which is equivalent to a single dose in adults, and inject subcutaneously. Immunization is carried out on days 0, 14, and 28 respectively. Whole blood is collected from the neck vein 42 days after immunization, and serum is separated. The specific test groups are shown in Table 2.

Table 2: Test groups

group	12F Conjugate Stock Solution Binding Process
PCV-1	Sodium periodate process combined with CRM197
PCV-2	CDAP process combined with CRM197
PCV-3	CDAP process combined with TT
PCV-4	PIA reducing amine method (example 1)
PCV-5	PIA-ADH method (example 2)
PCV-6	PIA-ADH method (example 3)
PCV-7	NCS reduced amine method (Pfizer)
PCV-8	NCS-ADH method (example 4)

ELISA method was used to detect the titer of IgG against various pneumococcal capsular polysaccharides, and the operation was performed according to the standardized WHO ELISA procedure. The immunogenicity of 12F polysaccharide conjugates is shown in FIG. 14 .

According to the standardized OPA procedure, the OPA GMT of the pooled serum was measured, and the immunogenicity of the 12F polysaccharide conjugate is shown in Figure 15.

Embodiment 12 NMR structure comparison

Comparison of the NMR structures of each relevant activation/derivative product in Examples 1 to 8

Activated polysaccharides obtained by oxidation of TEMPO-PIA;

Activated polysaccharides obtained by TEMPO-NCS oxidation;

Derivatized polysaccharide derived from ADH after oxidation of TEMPO-PIA;

Derivatized polysaccharide derived from ADH after TEMPO-NCS oxidation;

Activated polysaccharide obtained by oxidation of sodium periodate;

The NMR structures of the above activated products are shown in Figure 16, and the comparison of the NMR structures of each process (see Figures 21-22, where the polysaccharide numbers are shown in Table 4) shows that the 12F activated polysaccharides generated by TEMPO-PIA oxidation or TEMPO-NCS oxidation Similar to the structure of 12F capsular polysaccharide, the signal of sugar ring proton region and fingerprint region is basically consistent, which also shows that the oxidation of TEMPO-PIA or TEMPO-NCS will not destroy the sugar ring with adjacent hydroxyl structure. Compared with the structure of 12F capsular polysaccharide, the NMR of the activated polysaccharide generated by the oxidation of sodium periodate is significantly different, indicating that this method has a certain damage to the six-membered ring structure of the sugar (the sugar ring is adjacent to the hydroxyl group), which may be due to this The destruction of the structure caused the poor stability of the activated polysaccharide obtained by the sodium periodate method. Table 3 shows the comparison of the stability of polysaccharide conjugates obtained by different methods.

Table 3: Comparison of the stability of 12F conjugates prepared by different processes

The comparison of the NMR structures of the above ADH-derived products and activated products (see Figures 16, 21-22) shows that ADH-derived products can be obtained under suitable conditions through two different routes of TEMPO-PIA or TEMPO-NCS. The generated 12F activated capsular polysaccharide is similar in structure to the 12F capsular polysaccharide. For specific data, see the table below. The results show that the activated polysaccharide generated by TEMPO-PIA oxidation or TEMPO-NCS oxidation greatly improves the integrity of the sugar ring with the adjacent hydroxyl structure. good reservation.

In the embodiment, the polysaccharide activated by TEMPO-PIA or TEMPO-NCS is derivatized with ADH, and compared with the 12F capsular polysaccharide, the biggest difference lies in two shifts, as shown in the box in Figure 21 or 22 Shift interval, this shift is the ADH two groups of methylene characteristic peaks δ1.57, δ2.18 (Figure 20).

Theoretically, after the ADH characteristic peak is integrated, the ratio of the polysaccharide characteristic peak integral can be used to calculate the derivatization rate of the polysaccharide, but the method for calculating the derivatization rate by NMR integration is poor (if both amino groups of ADH react with the carboxyl group of the polysaccharide, the product meaningless to this process), so the derivation trend can only be calculated by nuclear magnetic method. According to the detection method of ADH in the 2020 edition of the Chinese Pharmacopoeia, the three general rules 3118, the derivation rate is obtained, as shown in Table 4. The variation trend is the same as that of NMR derivation rate.

Table 4 polysaccharide derivatization rate

Through the detection results of various properties of the polysaccharide protein conjugates finally obtained by the oxidation of PIA and NCS in the above examples, it can be found that the products obtained by PIA have similar immunogenicity compared with the products obtained by NCS, but the products obtained by PIA have Greater stability.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (16)

1. A glycoconjugate, characterized in that the glycoconjugate is obtained by oxidizing the primary hydroxyl group in the bacterial capsular polysaccharide and coupling it to a carrier protein directly or via a spacer group, wherein the bacterial Capsular polysaccharides include Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F , 23F or 33F capsular polysaccharide, the reactive site of the capsular polysaccharide includes β-D-GalpNAc, α-D-Galp or α-D-Glcp.
2. The glycoconjugate according to claim 1, characterized in that the primary hydroxyl group in the bacterial capsular polysaccharide is oxidized with a nitroxyl compound and an iodine oxidizing agent, preferably, the nitroxyl compound is 2,2,6,6- Tetramethyl-1-piperidinyloxy radical or a compound containing 2,2,6,6-tetramethyl-1-piperidinyloxy radical structure, more preferably, the iodine oxidizing agent is PIA.
3. The glycoconjugate according to claim 1, wherein the bacterial capsular polysaccharide is 12F type capsular polysaccharide.
4. The glycoconjugate according to claim 1, wherein the carrier protein is selected from the group consisting of diphtheria toxoid mutant, tetanus toxoid, outer membrane protein from Gram-negative bacteria, Haemophilus influenzae Surface lipoprotein, fusion protein of H. influenzae HiD protein gene and H. influenzae Hin47 protein gene, pertussis toxin, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein, or respiratory syncytial virus F and G proteins or active portions thereof.
5. The glycoconjugate according to claim 1, wherein the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3.
6. The glycoconjugate according to claim 1, characterized in that, for every 10 to 30 saccharide repeating units of the bacterial capsular polysaccharide, there is at least one between the carrier protein and the bacterial capsular polysaccharide covalent bond.
7. A method for preparing a glycoconjugate, characterized in that it comprises:

   Selectively oxidize the primary hydroxyl groups of bacterial capsular polysaccharides to aldehyde groups with nitroxyl compounds and iodine oxidants to obtain bacterial capsular polysaccharides containing aldehyde groups;

   Optionally, reacting the bacterial capsular polysaccharide containing an aldehyde group with a spacer to obtain a bacterial capsular polysaccharide derivative containing an aldehyde group;

   Prepare the glycoconjugate by reacting the aldehyde-containing bacterial capsular polysaccharide or the aldehyde-containing bacterial capsular polysaccharide derivative with the amino group or carboxyl group of the carrier protein, preferably, the aldehyde-containing bacterial capsular polysaccharide derivative and the carrier The protein is reacted in the presence of EDAC to prepare the glycoconjugate;

   Wherein, the bacterial capsular polysaccharide also includes Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, Capsular polysaccharides of type 19A, 19F, 20, 22F, 23F or 33F.
8. The method for preparing a glycoconjugate according to claim 7, wherein the iodine oxidizing agent is PIA.
9. A method for preparing a glycoconjugate according to claim 7 or 8, wherein the nitroxyl compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical Or a compound containing a 2,2,6,6-tetramethyl-1-piperidinyloxy radical structure.
10. The method for preparing a glycoconjugate according to claim 7, wherein the spacer is selected from ADH or CDH.
11. The preparation method of a kind of glycoconjugate according to claim 7, is characterized in that, described carrier protein is selected from diphtheria toxoid mutant, tetanus toxoid, outer membrane protein from Gram-negative bacteria , Haemophilus influenzae surface lipoprotein, fusion protein formed by Haemophilus influenzae HiD protein gene and Haemophilus influenzae Hin47 protein gene, pertussis toxin, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein Or respiratory syncytial virus F and G proteins or active parts thereof.
12. The preparation method of a glycoconjugate according to claim 7, characterized in that the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3.
13. The glycoconjugate prepared according to the preparation method described in any one of claims 7-12.
14. The immunogenic composition is characterized in that it comprises the glycoconjugate according to any one of claims 1-6 or the glycoconjugate according to claim 13, and pharmaceutically acceptable excipients, carriers and/or or thinner.
15. The immunogenic composition according to claim 14, wherein the composition comprises Streptococcus pneumoniae 12F capsular polysaccharide conjugate, and the Streptococcus pneumoniae 12F capsular polysaccharide conjugate is The nitroxyl compound and the iodine oxidizing agent selectively oxidize the primary hydroxyl group of the 12F type bacterial capsular polysaccharide to an aldehyde group, and obtain the 12F type bacterial capsular polysaccharide containing an aldehyde group;

    Optionally, reacting the 12F type bacterial capsular polysaccharide containing aldehyde group with the spacer to obtain the 12F type bacterial capsular polysaccharide derivative containing aldehyde group;

    Prepared by reacting the 12F type bacterial capsular polysaccharide containing aldehyde group or the 12F type bacterial capsular polysaccharide derivative containing aldehyde group with the amino group or carboxyl group of the carrier protein;

    Moreover, the composition also includes Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F , 20, 22F, 23F and 33F type capsular polysaccharide conjugates.
16. The glycoconjugate according to any one of claims 1-6, the glycoconjugate according to claim 13 or the immunogenic composition according to any one of claims 14-15 are used in the preparation of preventing and/or treating individual pneumonia Streptococcal infection, Streptococcus pneumoniae-associated drug or vaccine application.

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