RU2805417C1 - Capsular polysaccharides of streptococcus pneumoniae and their conjugates - Google Patents

Abstract

FIELD: polysaccharides.

SUBSTANCE: group of inventions is related to activated polysaccharides of Streptococcus pneumoniae serotypes 10A, 22F or 33F. An immunogenic conjugate containing polysaccharides of Streptococcus pneumoniae serotypes 10A, 22F or 33F covalently bound to a carrier protein, an immunogenic composition and a vaccine containing an immunogenic conjugate are proposed. The immunogenic conjugate contains a polysaccharide of Streptococcus pneumoniae serotype 10A, 22F or 33F, covalently linked to a carrier protein, and has a degree of conjugation from 2 to 15. The conjugate is prepared by making isolated capsular polysaccharide of serotype 10A, 22F, or 33F react with a periodate, quenching the oxidation reaction, mixing the activated polysaccharide with a carrier protein, and reducing it.

EFFECT: immunogenic conjugate, immunogenic composition and vaccine based thereon are suitable for manufacturing of a medicament for protecting a subject against Streptococcus pneumoniae infection.

27 cl, 6 dwg, 3 tbl, 5 ex

Description

Field of invention

This invention relates to activated polysaccharides of Streptococcus pneumoniae serotypes 10A, 22F or 33F and methods for their preparation. The invention also relates to immunogenic conjugates containing polysaccharides of Streptococcus pneumoniae serotypes 10A, 22F or 33F covalently linked to a carrier protein, methods for their preparation and immunogenic compositions and vaccines containing them.

Prior Art

Streptococcus pneumoniae are gram-positive, lanceolate cocci that are usually observed in pairs (diplococci), but also in short chains or as single cells. They grow well on blood agar plates to form shiny colonies and exhibit alpha hemolysis and when grown anaerobically they exhibit beta hemolysis. The cells of most pneumococcal serotypes have a capsule, which is a polysaccharide coating surrounding each cell. This capsule is a virulence determinant in humans as it interferes with phagocytosis by preventing antibodies from attaching to bacterial cells. There are currently more than 90 known identified pneumococcal capsular serotypes, with the 23 most common serotypes accounting for approximately 90% of invasive disease worldwide. As a vaccine, pneumococcal polysaccharide coating can confer a reasonable degree of immunity to Streptococcus pneumonia in subjects with developed and uncompromised immune systems, but capsular polysaccharide conjugated to a carrier protein provides an immune response in young children and the elderly, who are also at high risk of infected with pneumococci.

Since the introduction of the first 7-valent pneumococcal conjugate vaccine (PCV7 or Prevenar) in 2000, invasive disease caused by these seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) has almost disappeared. The addition of serotypes 1, 3, 5, 6A, 7F and 19A to Prevenar 13 further reduced the incidence of invasive pneumococcal disease.

None of the pneumococcal vaccines currently available on the market provide adequate protection against Streptococcus pneumoniae serotypes 10A, 22F, or 33F in humans and, in particular, in children under 2 years of age. Thus, there is a need for immunogenic compositions that can be used to induce an immune response against Streptococcus pneumonia serotypes 10A, 22F or 33F.

Summary of the invention

In one aspect of the present invention, there is provided a method for producing activated capsular polysaccharide of Streptococcus pneumoniae serotypes 10A, 22F or 33F, comprising the following steps:

(a) interaction of the isolated capsular polysaccharide of serotypes 10A, 22F or 33F with an oxidizing agent; And;

(b) quenching the oxidation reaction by adding a quenching agent, resulting in an activated polysaccharide of Streptococcus pneumoniae serotypes 10A, 22F or 33F.

In yet another aspect, the invention relates to an activated capsular polysaccharide of Streptococcus pneumoniae serotypes 10A, 22F or 33F, obtained or obtained by the activation method disclosed in this description of the invention.

In another aspect of the present invention, there is provided a method for producing an immunogenic conjugate containing a polysaccharide of Streptococcus pneumoniae serotypes 10A, 22F or 33F covalently linked to a carrier protein, comprising the following steps:

(a) mixing an activated polysaccharide of serotypes 10A, 22F or 33F obtained or obtained by the method disclosed in this description of the invention with a carrier protein; And,

(b) interaction of a mixture of an activated polysaccharide of serotype 10A, 22F or 33F and a carrier protein with a reducing agent to form a polysaccharide of serotype 10A, 22F or 33F:carrier protein conjugate.

In yet another aspect, the present invention provides a serotype 10A, 22F, or 33F polysaccharide:carrier protein conjugate produced or produced by the method described herein.

In yet another aspect of the present invention, immunogenic compositions and vaccines are provided containing the immunogenic conjugate disclosed herein.

In yet another aspect, the present invention provides a method of treating or preventing Streptococcus pneumoniae infection, disease or condition associated with Streptococcus pneumoniae serotype 10A, 22F or 33F in a subject, comprising the step of administering a therapeutically or prophylactically effective amount of an immunogenic composition or vaccine disclosed herein. description of the invention.

Brief description of graphic materials

In FIG. Figure 1 shows the structure of the capsular repeating unit polysaccharideStreptococcus pneumoniae serotype 10A.

In FIG. Figure 2 shows the structure of the capsular repeating unit. polysaccharideStreptococcus pneumoniae serotype 22F.

In FIG. Figure 3 shows the structure of the capsular repeating unit. polysaccharideStreptococcus pneumoniae serotype 33F.

In FIG. 4 shows the molecular weight of activated polysaccharide serotype 22F as a function of the amount of oxidizing agent at 23°C with or without a quenching step.

In FIG. 5 shows the molecular weight of activated polysaccharide serotype 22F as a function of the amount of oxidizing agent at 4° C. with or without a quenching step.

In FIG. 6 shows the molecular weight of the activated polysaccharide serotype 33F as a function of the amount of oxidizing agent at 2-8°C with or without a quenching step or at 23°C without a quenching step.

Detailed Description of the Invention

The present invention will be better understood by reference to the following detailed description of preferred embodiments of the invention and to the examples included in this specification. Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one skilled in the art to which the invention relates. Although any methods and substances similar or equivalent to those described herein may be used in the practice or testing of the present invention, certain preferred methods and substances are described herein. The described embodiments and claims will use certain terminology as defined below.

When used herein, the singular forms include plural references unless otherwise indicated. Thus, for example, references to “method” include one or more methods and/or steps of the type described herein and/or that will become apparent to those skilled in the art upon reading this specification.

As used herein, the term “molecular weight” of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by gel filtration chromatography (SEC) coupled with a multi-angle laser light scattering (MALLS) detector.

As used herein, the term “degree of oxidation” (DO) refers to the number of sugar repeat units per aldehyde group formed upon activation of the isolated polysaccharide by an oxidizing agent. The oxidation state of the polysaccharide can be determined using conventional methods known to those skilled in the art.

It should be noted that as used herein, terms such as “comprises,” “contains,” “comprising,” “contains,” “comprising,” and the like may have the meaning ascribed to them under US patent law; For example, they can mean "includes", "included", "including" and the like. Such terms refer to the inclusion of specific ingredients or set of ingredients without excluding any other ingredients. Terms such as "essentially consisting of" and "essentially consisting of" have the meaning ascribed to them in US patent law, e.g. they allow the inclusion of additional ingredients or steps that do not detract from the novel or essential features of the invention, that is, they exclude additional undescribed ingredients or steps that detract from the novel or essential features of the invention. The terms “consisting of” and “consisting of” have the meanings ascribed to them in United States patent law; namely, that these terms are of a closed type. Accordingly, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.

As used herein, the term “conjugates” or “glycoconjugates” refers to a polysaccharide covalently conjugated to a carrier protein. The glycoconjugates of the invention and immunogenic compositions containing them may contain some free polysaccharide.

As used herein, the term “serotype 10A glycoconjugate” or “serotype 10A conjugate” refers to the isolated capsular polysaccharide of Streptococcus pneumoniae serotype 10A covalently conjugated to a carrier protein.

As used herein, the term “serotype 22F glycoconjugate” or “serotype 22F conjugate” refers to the isolated capsular polysaccharide of Streptococcus pneumoniae serotype 22F covalently conjugated to a carrier protein.

As used herein, the term “serotype 33F glycoconjugate” or “serotype 33F conjugate” refers to the isolated capsular polysaccharide of Streptococcus pneumoniae serotype 33F covalently conjugated to a carrier protein.

As used herein, the term “serotype 10A polysaccharide” refers to the capsular polysaccharide of Streptococcus pneumoniae serotype 10A.

As used herein, the term “serotype 22F polysaccharide” refers to the capsular polysaccharide of Streptococcus pneumoniae serotype 22F.

As used herein, the term “serotype 33F polysaccharide” refers to the capsular polysaccharide of Streptococcus pneumoniae serotype 33F.

As used herein, the term “free polysaccharide” means a capsular polysaccharide that is not covalently conjugated to a carrier protein but is nonetheless present in the capsular polysaccharide-carrier protein conjugate composition. The free polysaccharide may be non-covalently associated with (ie, non-covalently associated with, adsorbed to, or enclosed in or with) a polysaccharide-carrier protein conjugate.

The percentage of free polysaccharide is measured after final purification of the serotype 10A, 22F, or 33F capsular polysaccharide carrier protein conjugate. Preferably it is measured within 4 weeks after final clearance. It is expressed as a percentage of the total polysaccharide in the sample.

As used herein, “conjugate,” “conjugated,” and “conjugating” refer to the process by which the capsular polysaccharide of Streptococcus pneumoniae is covalently associated with a carrier protein.

The term "subject" refers to a mammal, including a human, or a bird, fish, reptile, amphibian or any other animal. The term "subject" further includes pets or research animals. Non-limiting examples of pets or research animals include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, monkeys, birds, snakes, lizards, fish, turtles and frogs. The term "subject" also includes farm animals. Non-limiting examples of farm animals include: alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese and turkeys.

In the production of multivalent conjugate pneumococcal vaccines aimed at preventing invasive disease caused by the organism Streptococcus pneumoniae (also known as pneumococcus), selected serotypes of Streptococcus pneumoniae are grown to provide the polysaccharides necessary to produce the vaccine. Cells are grown in fermenters with lysis induced at the end of fermentation by the addition of sodium deoxycholate or an alternative lysis agent. The lysate broth is then collected for subsequent purification and extraction of the capsular polysaccharide that surrounds the bacterial cells. Once activated and conjugated to a carrier protein, the polysaccharide is incorporated into the final vaccine product and provides immunity to selected Streptococcus pneumoniae serotypes in the vaccine's target population.

The immunogenicity of a polysaccharide:carrier protein conjugate depends on several factors, including the size of the polysaccharide. Polysaccharide size is a quality characteristic analyzed for each batch of the drug and should be properly controlled. The high molecular weight of capsular polysaccharides is capable of inducing certain antibody immune responses due to the higher valency of the epitopes present on the antigenic surface. In general, it is preferable to prevent or limit undesirable reduction in the size of the polysaccharide during conjugate preparation to maintain the immunogenicity of the polysaccharide. In addition, it is important to reduce batch-to-batch variability in polysaccharide molecular weight during the activation and subsequent conjugation steps to maintain consistency in conjugate performance.

Reductive amination chemistry (RAC) has been shown by the inventors to be a suitable method for preparing a carrier protein-capsular polysaccharide conjugate of Streptococcus pneumoniae serotype 10A, 22F or 33F. The RAC approach involves activation of the polysaccharide by oxidation and subsequent conjugation of the activated polysaccharide to a carrier protein by reduction. Polysaccharides serotypes 10A, 33F and 22F have proven to be particularly sensitive polysaccharides and are susceptible to degradation and size reduction during the oxidation step of conjugate preparation. In addition, by applying the hitherto known activation method, activated polysaccharide of serotypes 10A, 33F and 22F with different molecular weights is obtained.

Thus, there is a significant need for a method for producing activated polysaccharidesStreptococcus pneumoniaeserotype 10A, 22F or 33F, having a controlled and stable molecular weight.

The subject of the present invention is a method for producing activated polysaccharides of Streptococcus pneumoniae serotype 10A, 22F or 33. In particular, the subject of the invention is a method for producing activated polysaccharide of Streptococcus pneumoniae serotype 10A, 22F or 33F, comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 10A, 22F or 33F with an oxidizing agent; And

(b) quenching the oxidation reaction by adding a quenching agent, resulting in an activated polysaccharide of Streptococcus pneumoniae serotype 10A, 22F or 33F.

Isolation and purification of polysaccharide serotype 10A, 22F or 33F

The structures of polysaccharides of serotypes 10A, 22F and 33F are described in the literature (see, for example, Kamerling J P C. Pneumococcal polysaccharides: a chemical view. In: Tomasz A, editor.Streptococcus pneumoniae, molecular biology and mechanisms of disease. New York, N.Y: Mary Ann Liebert, Inc.; 2000. pp. 81-114).

As shown in FIG. 1, the polysaccharide repeating unit of serotype 22F consists of a branched pentasaccharide backbone (one glucuronic acid (GlcpA), one glucopyranose (Glcp) and one galactofuranose (Galf) and two rhamnopyranoses (Rhap)) with an αGlcp site connected to the C3-hydroxyl group of βRhap 45 Approximately 80% of the C2-hydroxyl groups of the βRhap residue in the polysaccharide repeating unit are O-acetylated.

As shown in FIG. 2, the repeating unit of the serotype 10A polysaccharide consists of two D-galactofuranoses, three D-galactopyranoses, one N-acetylgalactosamine and a phosphate bond connected to the saccharide chain through one D-ribitol.

As shown in FIG. 3, the repeating unit of the serotype 33F polysaccharide consists of three D-galactopyranoses, two D-galactofuranoses and one D-glucopyranose. It should be noted that the 5-D-galactofuranosyl residues are O -acetylated in approximately 90% of the repeating units of the 33F polysaccharide.

Capsular polysaccharides serotypes 10A, 22F and 33F can be obtained directly from bacteria using isolation methods known to those skilled in the art (see, for example, the methods described in US Patent Application Publications 20060228380, 20060228381, 20070184071, 20070184072, 20070231 340 and 20080102498 or WO2008118752). Additionally, they can be produced using synthesis protocols.

Strains of Streptococcus pneumoniae serotypes 10A, 22F and 33F used to obtain the corresponding polysaccharides that are used in the immunogenic conjugates of the invention can be obtained from established culture collections or clinical samples.

Purified serotype 10A, 22F and 33F polysaccharides are prepared using methods well known to those skilled in the art. The bacterial cells are preferably grown in a soy-based medium. After fermentation of bacterial cells that produce capsular polysaccharides of Streptococcus pneumoniae serotype 10A, 22F or 33F, the bacterial cells are lysed to obtain a cell lysate. Bacterial cells can be lysed using any lysis agent. A "lysing agent" is any agent that helps break down the cell membrane and release autolysin, which causes cell lysis, including, for example, detergents. As used herein, the term "detergent" refers to an anionic or cationic detergent capable of inducing lysis of bacterial cells. Typical examples of such detergents for use in the methods of the present invention include sodium deoxycholate (DOC), N-lauroyl sarcosine, sodium chenodeoxycholic acid and saponins.

In one embodiment of the present invention, the lysis agent used to lyse bacterial cells is DOC. DOC is the sodium salt of the bile deoxycholic acid, which is usually obtained from biological sources such as cows or oxen. DOC activates the LytA protein, which is an autolysin involved in cell wall growth and division in Streptococcus pneumoniae . The LytA protein has choline-binding domains in its C-terminal portion, and mutations of the lytA gene are known to produce LytA mutants that are resistant to DOC-induced lysis.

In one embodiment of the present invention, the lysis agent used to lyse bacterial cells is a non-animal lysis agent. Non-animal lysing agents for use in the methods of the present invention include agents from non-animal sources with a mechanism of action similar to DOC (ie, that interfere with LytA function and result in lysis of Streptococcus pneumoniae cells). Such non-animal lysing agents include, but are not limited to, DOC analogs, surfactants, detergents, and structural choline analogs. In one embodiment, the non-animal lysing agent is selected from the group consisting of decane sulfonic acid, tert- octylphenoxy-poly(oxyethylene) ethanols (eg Igepal® CA-630, CAS#: 9002-93-1, available from Sigma Aldrich, St. Louis , MO), octylphenol ethylene oxide condensates (e.g. Triton® X-100, available from Sigma Aldrich, St. Louis, MO), sodium N-lauroyl sarcosine, lauryl iminodipropionate, sodium dodecyl sulfate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate and cholate . In another embodiment, the non-animal lysing agent is N-lauroylsarcosine. In another embodiment, the lysis agent is sodium N-lauroyl sarcosine.

Capsular polysaccharides can then be purified from the cell lysate using purification methods known in the art, including the use of centrifugation, depth filtration, sedimentation, ultrafiltration, activated carbon treatment, diafiltration and/or column chromatography (see, e.g. , US Patent Application Publications 20060228380, 20060228381, 20070184071, 20070184072, 20070231340 and 20080102498 or WO2008118752). Purified serotype 10A, 22F or 33F polysaccharides can then be used to prepare immunogenic conjugates.

Preferably, in order to obtain serotype 22F conjugates or serotype 33F conjugates with suitable filterability and/or yield characteristics, the polysaccharides are adjusted to a lower molecular weight (MW) range prior to conjugation to the carrier protein. Preferably, the size of the purified serotype 22F polysaccharide or serotype 33F polysaccharide is reduced while maintaining critical characteristics of the polysaccharide structure, such as, for example, the presence of O-acetyl groups. Preferably, the size of the purified serotype 22F polysaccharide or serotype 33F polysaccharide is reduced by mechanical homogenization.

In a preferred embodiment, the size of the purified polysaccharide is reduced by high pressure homogenization. High pressure homogenization achieves high shear rates by introducing the process stream through a flow channel of sufficiently small dimensions. The shear rate is increased by using higher applied homogenization pressure, and the exposure time can be increased by recirculating the material flow through the homogenizer.

The high pressure homogenization process is particularly suitable for reducing the size of purified serotype 22F polysaccharide or serotype 33F polysaccharide while maintaining the structural characteristics of the polysaccharide, such as the presence of O-acetyl groups.

The isolated serotype 22F polysaccharide, obtained by purifying the serotype 22F capsular polysaccharide from a lysate of Streptococcus pneumoniae and optionally adjusting the purified polysaccharide to the desired size, can be characterized by various parameters, including, for example, molecular weight and mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the size-separated serotype 22F polysaccharide has a molecular weight in the range of 400 to 700 kDa.

An isolated serotype 33F polysaccharide obtained by purifying the capsular polysaccharide from a lysate of Streptococcus pneumoniae serotype 33F and optionally adjusting the purified polysaccharide to size can be characterized by various parameters, including, for example, molecular weight and mM acetate per mM serotype 33F polysaccharide.

The degree of O-acetylation of a polysaccharide can be determined by any method known in the art, for example by proton NMR (Lemercinier and Jones (1996) Carbohydrate Research 296; 83-96, Jones and Lemercinier (2002) J. Pharmaceutical and Biomedical Analysis 30; 1233-1247, WO 05/033148 or WO00/56357). Another commonly used method is described in Hestrin (1949) J. Biol. Chem. 180; 249-261. Preferably, the presence of O-acetyl groups is determined by ion HPLC (high performance liquid chromatography) analysis.

In a preferred embodiment, the isolated serotype 22F polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 22F;

- lysis of bacterial cells in the specified fermentation culture; And

- purification of polysaccharide serotype 22F from fermentation culture.

In a preferred embodiment, the isolated serotype 22F polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 22F;

- lysis of bacterial cells in the specified fermentation culture;

- purification of polysaccharide serotype 22F from fermentation culture; And,

- bringing the serotype 22F polysaccharide to size by mechanical bringing to size, preferably by high pressure homogenization.

In a preferred embodiment, the isolated serotype 22F polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 22F;

- lysis of bacterial cells in the specified fermentation culture;

- purification of polysaccharide serotype 22F from fermentation culture; And

- bringing the serotype 22F polysaccharide to size by mechanical bringing to size, preferably by high pressure homogenization,

wherein the isolated serotype 22F polysaccharide has a molecular weight ranging from 400 to 700 kDa.

In a preferred embodiment, the isolated serotype 33F polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 33F;

- lysis of bacterial cells in the specified fermentation culture; And

- purification of polysaccharide serotype 33F from fermentation culture.

In a preferred embodiment, the isolated serotype 33F polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 33F;

- lysis of bacterial cells in the specified fermentation culture;

- purification of polysaccharide serotype 33F from fermentation culture; And

- bringing the serotype 33F polysaccharide to size by mechanical bringing to size, preferably by high pressure homogenization.

The isolated serotype 10A polysaccharide obtained by purifying a lysate of the capsular polysaccharide of Streptococcus pneumoniae serotype 10A and optionally adjusting the purified polysaccharide to the desired size can be characterized by various parameters, including, for example, molecular weight. Preferably, the isolated serotype 10A polysaccharide is not adjusted to the desired size

In a preferred embodiment, the isolated serotype 10A polysaccharide is prepared by a process comprising the following steps:

- obtaining a fermentation culture of bacterial cells of Streptococcus pneumonia serotype 10A;

- lysis of bacterial cells in the specified fermentation culture; And

- purification of polysaccharide serotype 10A from fermentation culture.

Activation of polysaccharide serotype 10A, 22F or 33F

The isolated polysaccharide of serotype 10A, 22F or 33F is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 10A, 22F or 33F with an oxidizing agent; And

(b) quenching the oxidation reaction by adding a quenching agent, resulting in an activated polysaccharide of Streptococcus pneumoniae serotype 10A, 22F or 33F.

In a preferred embodiment, the concentration of the serotype 10A, 22F, 33F polysaccharide in step (a) is from 0.1 to 10 mg/ml. In a preferred embodiment, the concentration of the serotype 10A, 22F, 33F polysaccharide in step (a) is from 0.5 to 5 mg/ml. In a preferred embodiment, the concentration of the serotype 10A, 22F, 33F polysaccharide in step (a) is from 1 to 3 mg/ml. In a preferred embodiment, the concentration of the serotype 22F or 33F polysaccharide in step (a) is about 2 mg/ml. In a preferred embodiment, the concentration of the serotype 10A polysaccharide in step (a) is approximately 2.5 mg/ml.

In a preferred embodiment, the oxidizing agent is a periodate. Periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups and causes cleavage of the C-C bond. The term “periodate” includes both periodate and periodic acid. This term also includes both metaperiodate (IO ₄ ^- ) and orthoperiodate (IO ₆ ^5- ). The term “periodate” also includes various periodate salts, including sodium periodate and potassium periodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In a preferred embodiment, the periodate used to oxidize the serotype 10A, 22F, 33F polysaccharide is a metaperiodate. In a preferred embodiment, the periodate used to oxidize the serotype 10A, 22F, 33F polysaccharide is sodium metaperiodate.

In a preferred embodiment, the polysaccharide reacts with 0.01-10, 0.05-5, 0.1-1, 0.5-1, 0.7-0.8, 0.01-0.2, 0.05- 0.5, 0.05-0.2, 0.1-0.3 molar equivalents of oxidizing agent. In a preferred embodiment, the polysaccharide reacts with about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55 , 0.6, 0.65, 0.7, 0.75, 0.8. In a preferred embodiment, the polysaccharide reacts with about 0.10 molar equivalent of an oxidizing agent. In a preferred embodiment, the polysaccharide reacts with about 0.15 molar equivalent of an oxidizing agent. In a preferred embodiment, the polysaccharide reacts with about 0.25 molar equivalent of an oxidizing agent. In a preferred embodiment, the polysaccharide is reacted with about 0.5 molar equivalent of an oxidizing agent. In a preferred embodiment, the polysaccharide reacts with about 0.8 molar equivalent of an oxidizing agent.

In a preferred embodiment, the reaction time in step (a) is 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 10, 10 to 50, 10 to 40 , from 10 to 30, from 10 to 20 hours. In a preferred embodiment, when the polysaccharide is a serotype 10A polysaccharide, the reaction time in step (a) is from 1 to 10 hours. In a preferred embodiment, when the polysaccharide is a serotype 10A polysaccharide, the reaction time in step (a) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours. In a preferred embodiment, when the polysaccharide is a serotype 10A polysaccharide, the reaction time in step (a) is approximately 4 hours. In a preferred embodiment, when the polysaccharide is a serotype 22F polysaccharide, the reaction time in step (a) is from 10 to 20 hours. In a preferred embodiment, when the polysaccharide is a serotype 22F polysaccharide, the reaction time in step (a) is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours. In a preferred embodiment, when the polysaccharide is a serotype 33F polysaccharide, the reaction time in step (a) is from 15 to 25 hours. In a preferred embodiment, when the polysaccharide is a serotype 33F polysaccharide, the reaction time in step (a) is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 hours. In a preferred embodiment, when the polysaccharide is a serotype 33F polysaccharide, the reaction time in step (a) is approximately 20 hours.

In a preferred embodiment, the reaction temperature in step (a) is maintained at 1 to 30°C, 1 to 10°C, 10 to 20°C, 20 to 30°C, 2 to 8°C. In a preferred embodiment, the reaction temperature in step (a) is maintained at approximately 5°C.

In a preferred embodiment, step (a) is carried out in a buffer selected from sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) or Bis-Tris. In a preferred embodiment, step (a) is carried out in potassium phosphate buffer. In a preferred embodiment, step (a) is carried out in sodium phosphate buffer.

In a preferred embodiment, the buffer concentration is from 1 to 500 mM, from 1 to 300 mM, from 50 to 200 mM. In a preferred embodiment, the buffer concentration is approximately 100 mM.

In a preferred embodiment, the pH value in step (a) is 4 to 8, 5 to 7, 5.5 to 6.5. In a preferred embodiment, the pH value in step (a) is about 6. In a preferred embodiment, the pH value in step (a) is about 5.8.

In one embodiment, the quenching agent is selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphoric acid.

In one embodiment, the quenching agent is a 1,2-amino alcohol of the formula

(I),

where R ¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium and potassium salts of sulfite, bisulfite, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphoric acid.

In one embodiment, the quenching agent is an amino acid. In one embodiment, said amino acid is selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine and histidine.

In one embodiment, the quenching agent is a sulfite, such as bisulfate, dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound containing two vicinal hydroxyl groups (vicinal diols), that is, two hydroxyl groups covalently bonded to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II)

(II),

wherein each of R ¹ and R ² is independently selected from H, methyl, ethyl, propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid. In a preferred embodiment, the quenching agent is butane-1,2-diol.

In a preferred embodiment, the oxidizing agent is neutralized by adding 0.1 to 10, 0.5 to 5, 0.5 to 3, 0.5 to 2 molar equivalents of quenching agent. In a preferred embodiment, the oxidizing agent is neutralized by adding about 0.5, 1, 1.5, 2, 2.5, 3 molar equivalents of quenching agent. In a preferred embodiment, the oxidizing agent is neutralized by adding about 2 molar equivalents of a quenching agent.

In a preferred embodiment, the duration of step (b) is from 0.1 to 10, from 0.5 to 5 or from 0.5 to 2 hours. In a preferred embodiment, the duration of step (b) is about 0.5, 1, 1.5, 2.5 or 3 hours.

In a preferred embodiment, the reaction temperature in step (b) is maintained at 1 to 30°C, 1 to 25°C, 1 to 20°C, 1 to 10°C, 10 to 20°C, 2 to 8 °C. In a preferred embodiment, the reaction temperature in step (b) is maintained at about 1, 2, 3, 4, 5, 6, 7 or 8°C.

In a preferred embodiment, the pH value in step (b) is 4 to 8, 5 to 7, 5.5 to 6.5. In a preferred embodiment, the pH in step (b) is about 6.

In a preferred embodiment, the activated polysaccharide serotype 10A, 22F, 33F is purified. Activated polysaccharide serotype 10A, 22F, 33F can be purified by methods known to those skilled in the art, such as gel permeation chromatography (GPC), dialysis or ultrafiltration/diafiltration. For example, the activated polysaccharide is purified by concentration and diafiltration using an ultrafiltration device.

In a preferred embodiment, the isolated serotype 10A polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated serotype 10A polysaccharide with periodate; And

(b) quenching the oxidation reaction by adding butane-2,3-diol, resulting in an activated polysaccharide of serotype 10A.

In a preferred embodiment, the isolated serotype 10A polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 10A with periodate at a temperature of 2 to 8°C; And

(b) quenching the oxidation reaction by adding butane-2,3-diol at a temperature of 2 to 8°C, resulting in an activated polysaccharide of serotype 10A.

In a preferred embodiment, the isolated serotype 10A polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 10A with 0.2-0.3 molar equivalent of periodate at a temperature of 2 to 8°C; And

(b) quenching the oxidation reaction by adding 0.5 to 2 molar equivalents of butane-2,3-diol at 2 to 8°C, resulting in an activated serotype 10A polysaccharide.

In a preferred embodiment, the isolated serotype 22F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 22F with periodate; And

(b) quenching the oxidation reaction by adding butane-2,3-diol, resulting in an activated polysaccharide of serotype 22F.

In a preferred embodiment, the isolated serotype 22F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 22F with periodate at a temperature of 2 to 8°C; And

(b) quenching the oxidation reaction by adding butane-2,3-diol at a temperature of 2 to 8°C, resulting in an activated polysaccharide of serotype 22F.

In a preferred embodiment, the isolated serotype 22F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 22F with 0.05-0.2 molar equivalent of periodate at a temperature of 2 to 8°C; And

(b) quenching the oxidation reaction by adding 2-3, preferably 2 molar equivalents of butane-2,3-diol at a temperature of 2 to 8°C, resulting in an activated polysaccharide of serotype 22F.

In a preferred embodiment, the isolated serotype 33F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated serotype 33F polysaccharide with periodate; And,

(b) quenching the oxidation reaction by adding butane-2,3-diol, resulting in an activated polysaccharide of serotype 33F.

In a preferred embodiment, the isolated serotype 33F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 33F with periodate at a temperature of 2 to 8°C; And,

(b) quenching the oxidation reaction by adding butane-2,3-diol at a temperature of 2 to 8°C, resulting in an activated polysaccharide of serotype 33F.

In a preferred embodiment, the isolated serotype 33F polysaccharide is activated by a method comprising the following steps:

(a) interaction of the isolated polysaccharide of serotype 33F with 0.05-0.2 molar equivalent of periodate at a temperature of 2 to 8°C; And

(b) quenching the oxidation reaction by adding 0.5 to 1.5 molar equivalents of butane-2,3-diol at 2 to 8°C, resulting in an activated serotype 33F polysaccharide.

In a preferred embodiment, the invention relates to an activated capsular polysaccharide of serotype 10A obtained or obtained by the above described method.

In a preferred embodiment, the invention relates to an activated capsular polysaccharide of serotype 22F obtained or obtained by the above described method.

In a preferred embodiment, the invention relates to an activated capsular polysaccharide of serotype 33F obtained or obtained by the above described method.

In a preferred embodiment, the activated serotype 10A polysaccharide obtained in step (b) has a molecular weight of 20 to 100%, 30 to 95%, 40 to 95%, 60 to 95%, 85 to 95%, or about 90% of the molecular weight of the isolated polysaccharide used in step (a).

In a preferred embodiment, the activated serotype 10A polysaccharide obtained in step (b) has a molecular weight that is at least 20, 25, 30, 35, 40 or 45% of the molecular weight of the isolated polysaccharide used in step (a). In a preferred embodiment, the activated serotype 10A polysaccharide obtained in step (b) has a molecular weight of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98.99% molecular weight of the isolated polysaccharide used in step (a).

In a preferred embodiment, the activated serotype 22F polysaccharide obtained in step (b) has a molecular weight of 50 to 100%, 60 to 95%, 85 to 95%, or about 90% of the molecular weight of the isolated polysaccharide used in step (a ).

In a preferred embodiment, the activated serotype 22F polysaccharide obtained in step (b) has a molecular weight of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% molecular weight of the isolated polysaccharide used in step (a).

In a preferred embodiment, the activated serotype 33F polysaccharide produced in step (b) has a molecular weight of 50 to 100%, 60 to 95%, 85 to 95%, or about 90% of the molecular weight of the isolated polysaccharide used in step (a ).

In a preferred embodiment, the activated serotype 33F polysaccharide obtained in step (b) has a molecular weight of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% molecular weight of the isolated polysaccharide used in step (a).

In a preferred embodiment, the oxidation state of the activated polysaccharide serotype 10A, 22F, 33F is from 2 to 30, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 2 to 5, from 5 to 30, from 5 to 25, from 5 to 20, from 5 to 15, from 5 to 10, from 10 to 30, from 10 to 25, from 10 to 20, from 10 to 15, from 5 to 25, from 10 to 30, from 15 to 30, 15 to 25, 15 to 20, 20 to 30, 20 to 25. In a preferred embodiment, the oxidation state of the activated polysaccharide serotype 10A, 22F, 33F is 2 to 10, 4 to 8, 4 to 6, from 6 to 8, from 6 to 12, from 8 to 14, from 9 to 11, from 10 to 16, from 12 to 16, from 14 to 18, from 16 to 20, from 16 to 18, from 18 to 22, from 18 to 20.

In a preferred embodiment, the activated serotype 10A polysaccharide has a molecular weight of 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 100 to 300, 100 to 250, or 100 to 200 kDa . In a preferred embodiment, the serotype 10A activated polysaccharide has a molecular weight of 50 to 300 kDa. In a preferred embodiment, the serotype 10A activated polysaccharide has a molecular weight of 100 to 200 kDa. In a preferred embodiment, the serotype 10A activated polysaccharide has a molecular weight of 100 to 200 kDa and an oxidation number of 5 to 20, 5 to 15, 8 to 14, 8 to 12, or 9 to 11. In a preferred embodiment, the serotype 10A activated polysaccharide has a molecular weight of 100 to 200 kDa and an oxidation number of 9 to 11.

In a preferred embodiment, the activated serotype 22F polysaccharide has a molecular weight of 25 to 1000, 100 to 1000, 300 to 800, 300 to 700, 300 to 600, 400 to 1000, 400 to 800, 400 to 700, or from 400 to 600 kDa. In a preferred embodiment, the serotype 22F activated polysaccharide has a molecular weight of 300 to 800 kDa. In a preferred embodiment, the serotype 22F activated polysaccharide has a molecular weight of 400 to 800 kDa. In a preferred embodiment, the serotype 22F activated polysaccharide has a molecular weight of 400 to 600 kDa. In a preferred embodiment, the activated serotype 22F polysaccharide has a molecular weight of 400 to 800 kDa and an oxidation number of 10 to 25, 10 to 20, 12 to 20, or 14 to 18. In a preferred embodiment, the activated serotype 22F polysaccharide has a molecular weight of 400 up to 800 kDa and oxidation state from 10 to 20.

In a preferred embodiment, the activated serotype 22F polysaccharide contains at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mM acetate per mM serotype 22F polysaccharide . In a preferred embodiment, the activated serotype 22F polysaccharide contains at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the activated serotype 22F polysaccharide contains at least 0.6 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the activated serotype 22F polysaccharide contains at least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide has a molecular weight of 400 to 800 kDa and contains at least 0.6 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide has a molecular weight of 400 to 800 kDa, an oxidation state of 12 to 20, and contains at least 0.6 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide has a molecular weight of 50 to 1250, 200 to 1200, 500 to 1200, 500 to 1000, 700 to 1200, 800 to 1200, 800 to 1100, 900 to 1200, from 800 to 1000 kDa. In a preferred embodiment, the serotype 33F activated polysaccharide has a molecular weight of 500 to 1000 kDa. In another preferred embodiment, the serotype 33F activated polysaccharide has a molecular weight of 50 to 600, 50 to 500, or 50 to 400 kDa.

In a preferred embodiment, the serotype 33F activated polysaccharide contains at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 or about 0.9 mM acetate per mM polysaccharide serotype 33F. In a preferred embodiment, the activated serotype 33F polysaccharide contains at least 0.6, 0.7 or 0.8 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the activated serotype 33F polysaccharide contains at least 0.6 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the activated serotype 33F polysaccharide contains at least 0.7 mM acetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide has a molecular weight of 800 to 1200 kDa and contains at least 0.7 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the activated serotype 33F polysaccharide has a molecular weight of 800 to 1200 kDa, contains at least 0.7 mM acetate per mM serotype 33F polysaccharide, and has an oxidation state of 10 to 20.

In a preferred embodiment, the activated serotype 33F polysaccharide has a molecular weight of 50 to 200 kDa and contains at least 0.7 mM acetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide has a molecular weight of 50 to 200 kDa and contains at least 0.7 mM acetate per mM serotype 33F polysaccharide and has an oxidation state of 10 to 20.

In one embodiment, the activated polysaccharide serotype 10A, 22F, 33F is lyophilized, optionally in the presence of a cryoprotectant/lyoprotectant. In a preferred embodiment, the cryoprotectant/lyoprotectant is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinite. In a preferred embodiment, the cryoprotectant/lyoprotectant is sucrose. The lyophilized activated polysaccharide can then be mixed with a solution containing the carrier protein.

In another embodiment, the activated polysaccharide serotype 10A, 22F, 33F is mixed with a carrier protein and lyophilized, optionally in the presence of a cryoprotectant/lyoprotectant. In a preferred embodiment, the cryoprotectant/lyoprotectant is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinite. In a preferred embodiment, the cryoprotectant/lyoprotectant is sucrose. The co-lyophilized polysaccharide and carrier protein can then be resuspended in solution and reacted with a reducing agent.

In one embodiment, the invention relates to a lyophilized activated polysaccharide of serotype 10A. In one embodiment, the invention relates to a lyophilized activated polysaccharide of serotype 22F. In one embodiment, the invention relates to a lyophilized activated polysaccharide of serotype 33F.

In one embodiment, the invention relates to a co-lyophilized activated polysaccharide of serotype 10A and a carrier protein. In a preferred embodiment, the carrier protein is CRM ₁₉₇ . In one embodiment, the invention relates to a co-lyophilized activated serotype 22F polysaccharide and a carrier protein. In a preferred embodiment, the carrier protein is CRM ₁₉₇ . In one embodiment, the invention relates to a co-lyophilized activated serotype 33F polysaccharide and a carrier protein. In a preferred embodiment, the carrier protein is CRM ₁₉₇ .

Conjugation of activated polysaccharide serotypes 10A, 22F, 33F with a carrier protein

The activated polysaccharide serotype 10A, 22F, 33F disclosed herein can be conjugated to a carrier protein by a method comprising the following steps:

(a) mixing activated polysaccharide serotype 10A, 22F, 33F with a carrier protein; And

(b) interaction of a mixture of activated polysaccharide serotype 10A, 22F, 33F and carrier protein with a reducing agent to form a polysaccharide serotype 10A, 22F, 33F:carrier protein conjugate.

Conjugation of an activated serotype 22F or 33F polysaccharide to a carrier protein by reductive amination in dimethyl sulfoxide (DMSO) is useful for preserving the O-acetyl content of the polysaccharide compared to, for example, reductive amination in the aqueous phase, where the level of O-acetylation of the polysaccharide is significantly reduced. In a preferred embodiment, step (a) and step (b) are performed in DMSO.

In a preferred embodiment, step (a) comprises dissolving the lyophilized polysaccharide serotype 10A, 22F, 33F in an aqueous solution containing the carrier protein, or in a solution containing the carrier protein and DMSO. In a preferred embodiment, step (a) comprises dissolving the co-lyophilized serotype 10A, 22F, 33F polysaccharide and carrier protein in an aqueous solution or in DMSO.

When step (a) and (b) are performed in an aqueous solution, said solution contains a buffer, preferably selected from a buffer, preferably selected from PBS (phosphate buffered saline), MES (2-morpholinoethanesulfonic acid), HEPES (2-[4- (2-hydroxyethyl)piperazin-1-yl]-ethanesulfonic acid), Bis-tris, ADA (N-(2-acetamido)iminodiacetic acid), PIPES (1,4-piperazinediethanesulfonic acid), MOPSO (3-morpholino-2 -hydroxypropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), DIPSO (3-( N,N -bis[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-N ,N'-bis(2-hydroxypropanesulfonic acid)), TEA (triethylamine), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), Bicine or HEPB (N-(2-hydroxyethyl)piperazine-N'- (4-butanesulfonic acid)), at a pH of 6.0 to 8.5, 7 to 8, or 7 to 7.5. In a preferred embodiment, the buffer is PBS. In a preferred embodiment, the pH value is about 7.3.

In a preferred embodiment, the concentration of activated polysaccharide serotype 10A, 22F, 33F in step (a) is 0.1 to 10 mg/ml, 0.5 to 5 mg/ml, 0.5 to 2 mg/ml. In a preferred embodiment, the concentration of activated polysaccharide serotype 10A, 22F, 33F in step (a) is about 0.1, 0.2, 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, 2.1, 2 .2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 mg/ml.

In a preferred embodiment, the initial ratio (wt/wt) of activated polysaccharide serotype 10A, 22F, 33F to carrier protein is from 5:1 to 0.1:1, from 2:1 to 0.1:1, from 2:1 to 1:1, from 1.5:1 to 1:1, from 0.1:1 to 1:1, from 0.3:1 to 1:1, from 0.6:1 to 1:1.2. In a preferred embodiment, the initial ratio of activated serotype 10A, 22F, 33F polysaccharide to carrier protein is from about 0.6:1 to 1:1.2. In a preferred embodiment, the initial ratio of activated serotype 10A, 22F, 33F polysaccharide to carrier protein is about 0.4:1, 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.

In a preferred embodiment in step (b), the activated polysaccharide serotype 10A, 22F, 33F is reacted with 0.1-10 molar equivalents, 0.5-5 molar equivalents, 0.5-2.5 molar equivalents of a reducing agent. In a preferred embodiment, in step (b), the activated polysaccharide of serotype 10A, 22F or 33F reacts with about 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, 2.1, 2.2, 2.3, 2.4 or 2.5 molar reducing agent equivalents.

In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride in the presence of a Brønsted or Lewis acid, aminoboranes such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, tert -BuMe ⁱ PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-methylpyridine borane (PEMB). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

In a preferred embodiment, the duration of step (b) is from 1 to 50, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 5 to 25, 10 to 25, 15 to 25 hours. In a preferred embodiment, the duration of step (b) is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28 hours.

In a preferred embodiment, step (b) maintains the reaction temperature at 10 to 40°C, 15 to 30°C, 20 to 26°C, 21 to 25°C. In a preferred embodiment, in step (b) the reaction temperature is maintained at about 21, 22, 23, 24 or 25°C.

In a preferred embodiment, the method of producing an immunogenic conjugate containing a serotype 10A, 22F, 33F polysaccharide covalently linked to a carrier protein further includes the step (step c) of blocking the unreacted aldehyde (quenching) by adding NaBH ₄ .

In a preferred embodiment, in step (c), unreacted aldehydes were blocked by adding 0.1 to 10 molar equivalents, 0.5 to 5 molar equivalents, or 1 to 3 molar equivalents of NaBH ₄ . In a preferred embodiment, in step (c), unreacted aldehydes were blocked by adding about 2 molar equivalents of NaBH ₄ .

In a preferred embodiment, the duration of step (c) is from 0.1 to 10, from 0.5 to 5 or from 2 to 4 hours. In a preferred embodiment, the duration of step (c) is approximately 3 hours.

In a preferred embodiment, the reaction temperature in step (c) is maintained at 10 to 40°C, 15 to 30°C, or 20 to 26°C. In a preferred embodiment, the reaction temperature in step (c) is maintained at approximately 23°C.

After conjugation of a serotype 10A, 22F, 33F polysaccharide to a carrier protein, the polysaccharide-carrier protein conjugate can be purified (enriched in terms of the amount of polysaccharide-carrier protein conjugate) by various methods known to one skilled in the art. These methods include dialysis, concentration/diafiltration procedures, tangential flow filtration precipitation/elution, column chromatography (DEAE ((diethylaminoethyl)-dextran) or hydrophobic chromatography) and depth filtration.

In a preferred embodiment, the carrier protein is non-toxic and non-reactogenic and can be obtained in sufficient quantity and purity. Carrier proteins must be amenable to standard conjugation procedures.

In a preferred embodiment, the activated polysaccharide serotype 10A, 22F, 33F is conjugated to a carrier protein that is selected from the group consisting of: DT (diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, CRM197 (non-toxic but antigenically identical variant diphtheria toxin) other DT point mutants such as CRM176, CRM228, CRM 45 (Uchida et al J. BioI. Chem. 218; 3838-3844, 1973); CRM 9, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser, and/or Ala 158 to Gly, and other mutations described in US 4709017 or US 4950740; mutation of at least one or more residues of Lys 516, Lys 526, Phe 530 and/or Lys 534, and other mutations described in US 5917017 or US 6455673; or a fragment described in US 5843711, pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), including some detoxified ply (pneumolysin), for example dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX including PhtA, PhtB, PhtD, PhtE (PhtA, PhtB, PhtD or PhtE sequences are described in WO 00/37105 or WO 00/39299) and Pht protein fusions, e.g. PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334, WO 03/54007, W02009/000826), OMPC (meningococcal outer membrane protein - usually extracted from N. meningitidis serogroup B - EP0372501), PorB (from N. meningitidis), PD (Haemophilus influenza protein D - see for example EP 0594610 B) or their immunologically functional equivalents, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP 0471177), cytokines, lymphokines, growth factors and hormones (WO10 91/01146), artificial proteins containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824), such as N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7), pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B C difficile (WO 00/61761). In one embodiment, the activated polysaccharide serotype 10A, 22F, 33F is conjugated to DT (diphtheria toxoid). In another embodiment, the activated polysaccharide serotype 10A, 22F, 33F is conjugated to TT (tetanus toxoid). In another embodiment, the activated polysaccharide serotype 10A, 22F, 33F is conjugated to the C moiety of TT. In another embodiment, the activated polysaccharide serotype 10A, 22F, 33F is conjugated to PD (Haemophilus influenza protein D - see, for example, EP 0594610 B).

In a preferred embodiment, the serotype 10A, 22F, 33F activated polysaccharide is conjugated to a CRM ₁₉₇ protein. The CRM ₁₉₇ protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by diphtheria bacilli infected with the nontoxigenic phage β197 ^tox- , created by nitrosoguanidine mutagenesis of the toxigenic corinophage-beta (Uchida, T. et al. 1971, Nature New Biology 233:8-11). CRM ₁₉₇ was purified by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin, but differs from it by changing one base (guanine to adenine) in the structural gene. This single base change causes the amino acid glutamic acid to be replaced by glycine in the mature protein and eliminates the toxic properties of diphtheria toxin. CRM ₁₉₇ protein is a safe and effective T-cell-dependent saccharide carrier. More information about CMR ₁₉₇ and its receipt can be found, for example, in US 5614382.

In a preferred embodiment, the serotype 10A, 22F or 33F activated polysaccharide disclosed herein is conjugated to CRM ₁₉₇ by a method comprising the following steps:

(a) mixing activated polysaccharide serotype 10A, 22F or 33F with CRM ₁₉₇ ;

(b) reacting a mixture of activated polysaccharide serotype 10A, 22F or 33F and CRM ₁₉₇ with sodium cyanoborohydride to form a polysaccharide serotype 10A, 22F or 33F:CRM ₁₉₇ conjugate.

In a preferred embodiment, the serotype 10A, 22F or 33F activated polysaccharide disclosed herein is conjugated to CRM ₁₉₇ by a method comprising the following steps:

(a) mixing activated polysaccharide serotype 10A, 22F or 33F with CRM ₁₉₇ ;

(b) interaction of a mixture of activated polysaccharide serotype 10A, 22F or 33F and CRM ₁₉₇ with sodium cyanoborohydride to form a polysaccharide serotype 10A, 22F or 33F:CRM ₁₉₇ conjugate;

where steps (a) and (b) are performed in DMSO.

In a preferred embodiment, the serotype 10A, 22F or 33F activated polysaccharide disclosed herein is conjugated to CRM ₁₉₇ by a method comprising the following steps:

(a) mixing activated polysaccharide serotype 10A, 22F or 33F with CRM ₁₉₇ ;

(b) interaction of a mixture of activated polysaccharide serotype 10A, 22F or 33F and CRM ₁₉₇ with 0.5-2 molar equivalents of sodium cyanoborohydride to form a polysaccharide serotype 10A, 22F or 33F:CRM ₁₉₇ conjugate;

where steps (a) and (b) are performed in DMSO.

In one embodiment, the invention relates to an immunogenic conjugate obtained or obtained by the method disclosed in this description of the invention. In a preferred embodiment, the invention relates to an immunogenic conjugate prepared or produced by conjugating an activated polysaccharide of serotype 10A, 22F or 33F disclosed herein to a carrier protein by reductive amination. In a preferred embodiment, the invention relates to an immunogenic conjugate prepared or prepared by conjugating an activated polysaccharide of serotype 10A, 22F or 33F disclosed herein to a carrier protein by reductive amination in DMSO. In a preferred embodiment, the carrier protein is CRM ₁₉₇ .

Immunogenic conjugate serotype 10A

In a preferred embodiment, the serotype 10A immunogenic conjugate has a molecular weight of from 500 to 15,000; from 500 to 10000; from 2000 to 10000; or from 3000 to 8000 kDa. In a preferred embodiment, the serotype 10A immunogenic conjugate has a molecular weight of between 3000 and 8000 kDa. The molecular weight of the immunogenic conjugate is determined by SEC-MALLS.

In a preferred embodiment, the serotype 10A immunogenic conjugate contains less than about 50, 45, 40, 35, 30, 25, 20, or 15% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide. In a preferred embodiment, the serotype 10A immunogenic conjugate contains less than about 25% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide. In a preferred embodiment, the serotype 10A immunogenic conjugate contains less than about 20% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide. In a preferred embodiment, the serotype 10A immunogenic conjugate contains less than about 15% free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide.

In a preferred embodiment, the ratio (weight/weight) of serotype 10A polysaccharide to carrier protein in the conjugate is from 0.5 to 3. In a preferred embodiment, the ratio of serotype 10A polysaccharide to carrier protein in the conjugate is from 0.5 to 2, from 0. 5 to 1.5, 0.5 to 1, 1 to 1.5, 1 to 2. In a preferred embodiment, the ratio of serotype 10A polysaccharide to carrier protein in the conjugate is 0.8 to 1.4. In a preferred embodiment, the ratio of serotype 10A capsular polysaccharide to carrier protein in the conjugate is from 0.8 to 1.2.

Gel filtration chromatography medium (CL-4B) can be used to determine the relative molecular size distribution of the conjugate. Gel filtration chromatography (SEC) uses gravity-fed columns to profile the molecular size distribution of the conjugates. Large molecules forced out of the pores into the medium elute faster than small molecules. Fraction collectors are used to collect eluate from the column. Fractions are tested colorimetrically by saccharide analysis. To determine Kd, columns are calibrated to establish the fraction in which molecules are completely displaced (V ₀ ), (Kd=0) and the fraction representing maximum retention (V _i ), (Kd=1). The fraction in which a certain property of the sample is achieved (V _e ) is correlated with Kd using the expression Kd = (V _e -V ₀ )/ (V _i -V ₀ ).

In a preferred embodiment, at least 30% of the serotype 10A immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 40% of the immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 10A immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL column -4B. In a preferred embodiment, at least 60% of the serotype 10A immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, 50% to 80% of the serotype 10A immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column.

The degree of conjugation is determined by the number of lysine residues in the carrier protein that is conjugated to the polysaccharide of interest. Evidence of lysine modification in the carrier protein as a result of covalent bonds with polysaccharides is obtained through amino acid analysis using standard methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the CRM ₁₉₇ protein of the starting material used to produce the conjugated substances.

In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 5 to 15, from 5 to 10, from 8 to 15, from 8 to 12, from 10 to 15 or from 10 to 12. In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is from 6 to 8.

Immunogenic conjugate serotype 22F

In a preferred embodiment, the immunogenic conjugate contains at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7, or about 0.8 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.6 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the isolated polysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0 .85, .9, or .95. In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the isolated polysaccharide is at least 0.7. In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the isolated polysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the activated polysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0 .85, 0.9 or 0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the activated polysaccharide is at least 0.7. In a preferred embodiment, the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the activated polysaccharide is at least 0.9.

In a preferred embodiment, the serotype 22F immunogenic conjugate has a molecular weight of from 400 to 15,000; from 500 to 10000; from 2000 to 10000 kDa; from 3000 to 8000 kDa; or from 3000 to 5000 kDa. In a preferred embodiment, the serotype 22F immunogenic conjugate has a molecular weight of 3000 to 5000 kDa. The molecular weight of the immunogenic conjugate is measured by SEC-MALLS (multi-angle laser scattering detection gel chromatography).

In a preferred embodiment, the serotype 22F immunogenic conjugate contains less than about 50, 45, 40, 35, 30, 25, 20, or 15% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F immunogenic conjugate contains less than about 40% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F immunogenic conjugate contains less than about 25% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F immunogenic conjugate contains less than about 20% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide. In a preferred embodiment, the serotype 22F immunogenic conjugate contains less than about 15% free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide.

In a preferred embodiment, the ratio (weight/weight) of serotype 22F polysaccharide to carrier protein in the conjugate is from 0.5 to 3. In a preferred embodiment, the ratio of serotype 22F polysaccharide to carrier protein in the conjugate is from 0.5 to 2, from 0. 5 to 1.5, 0.8 to 1.2, 0.5 to 1, 1 to 1.5, or 1 to 2. In a preferred embodiment, the ratio of serotype 22F polysaccharide to carrier protein in the conjugate is from 0 .8 to 1.2. In a preferred embodiment, the ratio of serotype 22F capsular polysaccharide to carrier protein in the conjugate is from 0.9 to 1.1.

In a preferred embodiment, at least 30% of the serotype 22F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 40% of the immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 22F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL column -4B. In a preferred embodiment, at least 60% of the serotype 22F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, 50% to 80% of the serotype 22F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, 65% to 80% of the serotype 22F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column.

In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 4 to 7, from 5 to 15, from 5 to 10, from 8 to 15, from 8 to 12, 10 to 15, or 10 to 12. In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is from 4 to 7.

Immunogenic conjugate serotype 33F

In a preferred embodiment, the immunogenic conjugate contains at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.6 mM acetate per mM serotype 33F polysaccharide. In a preferred embodiment, the immunogenic conjugate contains at least 0.7 mM acetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the isolated polysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0 .85, 0.9 or 0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the isolated polysaccharide is at least 0.7. In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the isolated polysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the activated polysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0 .85, 0.9 or 0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the activated polysaccharide is at least 0.7. In a preferred embodiment, the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the activated polysaccharide is at least 0.9.

In a preferred embodiment, the serotype 3F immunogenic conjugate has a molecular weight of from 500 to 30,000; 500 to 25000; 500 to 20000; 500 to 15000; 500 to 10000; 1000 to 10000; 1000 to 8000; 1000 to 5000; 2000 to 10000 kDa; 2000 to 8000; or 2000 to 5000 kDa. In a preferred embodiment, the serotype 3F immunogenic conjugate has a molecular weight of 1000 to 5000 kDa. The molecular weight of the immunogenic conjugate is determined by SEC-MALLS.

In a preferred embodiment, the serotype 3F immunogenic conjugate contains less than about 50, 45, 40, 35, 30, 25, 20, or 15% free serotype 33F polysaccharide compared to the total amount of serotype 33F polysaccharide. In a preferred embodiment, the serotype 3F immunogenic conjugate contains less than about 25% free serotype 33F polysaccharide compared to the total amount of serotype 33F polysaccharide. In a preferred embodiment, the serotype 3F immunogenic conjugate contains less than about 20% free serotype 33F polysaccharide compared to the total amount of serotype 33F polysaccharide. In a preferred embodiment, the serotype 3F immunogenic conjugate contains less than about 15% free serotype 33F polysaccharide compared to the total amount of serotype 33F polysaccharide.

In a preferred embodiment, the ratio (weight/weight) of serotype 33F polysaccharide to carrier protein in the conjugate is from 0.4 to 3. In a preferred embodiment, the ratio of serotype 33F polysaccharide to carrier protein in the conjugate is from 0.5 to 2, from 0. 5 to 1.5, 0.5 to 1, 1 to 1.5, 1 to 2. In a preferred embodiment, the ratio of serotype 33F polysaccharide to carrier protein in the conjugate is from 0.5 to 1.5. In a preferred embodiment, the ratio of serotype 33F capsular polysaccharide to carrier protein in the conjugate is from 0.5 to 1.2.

In a preferred embodiment, at least 30% of the serotype 3F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 40% of the immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65% of the serotype 3F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, at least 60% of the serotype 3F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, from 40% to 80% of the serotype 3F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column. In a preferred embodiment, 45% to 65% of the serotype 3F immunogenic conjugate has a Kd value that is less than or equal to 0.3 on the CL-4B column.

In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is 1 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 5 to 15, from 5 to 10, from 8 to 15, from 8 to 12, from 10 to 15 or from 10 to 12. In a preferred embodiment, the degree of conjugation of the immunogenic conjugate is from 3 to 6.

Immunogenic composition

The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, wherein said composition can be used to elicit an immune response in a subject.

As used herein, "immunogenic" means the ability of an antigen (or an epitope of an antigen), such as a bacterial capsular polysaccharide or glycoconjugate, or an immunogenic composition containing an antigen, to elicit an immune response in a host, such as a mammal, either humorally or cellularly mediated. or both ways.

In a preferred embodiment, the immunogenic composition comprises a serotype 10A, 22F or 33F conjugate prepared by the method disclosed herein. In a preferred embodiment, an immunogenic composition containing a serotype 10A, 22F or 33F conjugate is prepared by the method disclosed herein.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 10A . In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 10A, as measured by a standard ELISA assay.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of killing S treptococcus pneumonia serotype 10A in the opsonophagocytic assay disclosed herein.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 22F . In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 22F, as measured by a standard ELISA assay.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of killing Streptococcus pneumonia serotype 22F in the opsonophagocytic assay disclosed herein.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 33F . In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of binding to S treptococcus pneumonia serotype 33F, as measured by a standard ELISA assay.

In one embodiment, the immunogenic composition disclosed herein, when administered to a subject, induces the formation of antibodies capable of killing S treptococcus pneumonia serotype 33F in the opsonophagocytic assay disclosed herein.

The formation of the immunogenic composition of the present invention can be accomplished using methods known in the art. For example, conjugates of serotype 10A, 22F or 33F can be formulated with a physiologically acceptable carrier to form a composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyhydric alcohols (eg, glycerin, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In a preferred embodiment, the immunogenic composition may contain at least one additional antigen. In a preferred embodiment, the immunogenic composition may contain at least one additional Streptococcus pneumoniae capsular polysaccharide.

In a preferred embodiment, the immunogenic composition may comprise at least one additional Streptococcus pneumoniae capsular polysaccharide conjugated to a carrier protein. In a preferred embodiment, said carrier protein is CRM ₁₉₇ .

In some embodiments, the immunogenic composition contains one or more adjuvants. As defined herein, an “adjuvant” is a substance that serves to increase the immunogenicity of the immunogenic composition of the invention. Thus, adjuvants help enhance the immune response and are well known to those skilled in the art. Suitable adjuvants to enhance the effectiveness of the composition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion compositions (with or without other specific immunostimulating agents, such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example,

(a) MF59 (PCT publication WO 90/14837) containing 5% squalene, 0.5%5 Tween 80 and 0.5% Span 85 (possibly containing varying amounts of MTP-PE (see below, although not required)) , prepared into submicron particles using a microfluidizer such as a Model 11OY microfluidizer (Microfluidics, Newton, MA),

(b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic-blocked polymer L121 and thr-MDP (see below), either microfluidized into a submicron emulsion or mixed to form an emulsion with larger particles, And

(c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL™), described in US Pat. No. 4,912,094 (Corixa), trehalose dimycolate (TDM) and cell wall scaffold (CWS), preferably MPL + CWS (Detox™);

(3) saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (US Pat. No. 5,057,540) or particles derived therefrom such as ISCOM (immune stimulating complexes) may be used;

(4) bacterial lipopolysaccharides, synthetic analogs of lipid A, such as aminoalkyl-glucosamine phosphate compounds (AGP) or derivatives or analogs thereof, which are available in Corixa, and which are described in US Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxotetradecanoylamino]ethyl 2-Deoxy-4-ophosphono-3-O-[(R)-3-tetradecanoyloxotetradecanoyl]-2-[(R)-3-tetradecanoyloxotetradecanoylamino ]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529), which is prepared in aqueous form or as a stable emulsion, synthetic polynucleotides such as oligonucleotides containing CpG motif(s) (US Pat. No. 6,207,646);

(5) cytokines such as interleukins (for example IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. ), interferons (eg interferon gamma), granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules 87-1 and 87-2, etc. .;

(6) detoxified mutants of a bacterial ADP-ribosylating toxin, such as cholera toxin (CT), either wild type or in mutant form, for example, where glutamic acid at amino acid position 29 is replaced by another amino acid, preferably histidine, according to the international application patent published as international publication WO 00/18434 (see also WO 02/098368 and WO 02/098369), pertussis toxin (PT) or E. coli heat labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, for example, WO 93/13302 and WO 92/19265); And

(7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-L-normuramyl-alanine-2-(1'-2'-dipalmitoyl- sn -glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In one embodiment of the present invention, the immunogenic compositions disclosed herein contain a CpG oligonucleotide as an adjuvant. CpG oligonucleotide, as used herein, refers to an immunostimulatory CpG oligodeoxynucleotide (CpG-ODN) and accordingly, these terms are used interchangeably unless otherwise noted. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs, which are unmethylated cytosine-guanine dinucleotides, possibly surrounded by certain preferred bases. The methylation status of the immunostimulatory CpG motif usually refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide that contains a 5' unmethylated cytosine linked by a phosphate bond to a 3' guanine and which activates the immune system through binding to Toll-like receptor 9 (TLR- 9). In another embodiment, the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides that activate the immune system through TLR9, but not as strongly as if the CpG motif(s) were/were unmethylated. Immunostimulatory CpG oligonucleotides may contain one or more palindromes, which in turn may surround the CpG dinucleotide. CpG oligonucleotides have been described in a number of issued patents, published patent applications and other publications, including US Pat. No. 6,194,388; 6207646; 6214806; 6218371; 6239116; and 6339068.

In one embodiment of the present invention, the immunogenic compositions disclosed herein contain any CpG oligonucleotide described on p. 3 (line 22) - p. 12 (line 36) in WO2010/125480.

Different classes of immunostimulatory CpG oligonucleotides have been identified. These are referred to as A, B, C and P class and are described in more detail on p. 3 (line 22) - p. 12 (line 36) in WO2010/125480. The methods of the invention encompass the use of these different classes of immunostimulatory CpG oligonucleotides.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a Class A CpG oligonucleotide. Preferably, the "Class A" CpG oligonucleotide of the invention has the following nucleic acid sequence: 5' GGGGACGACGTCGTGGGGGGG 3' (SEQ ID NO: 1). Some non-limiting examples of A-class oligonucleotides include:
5' G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3' (SEQ ID NO: 2) ; where * refers to the phosphorothioate bond and _ refers to the phosphodiester bond.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a Class B CpG oligonucleotide. In one embodiment, the CpG oligonucleotide for use in the present invention is a Class B CpG oligonucleotide represented by at least the formula:

5' X ₁ X ₂ CGX ₃ X ₄ 3', where X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. In one embodiment, X ₂ is adenine, guanine or thymine. In another embodiment, X ₃ is cytosine, adenine or thymine.

The sequences of the class B CpG oligonucleotides of the invention are those described in detail above in US patents 6194388, 6207646, 6214806, 6218371, 6239116 and 6339068. Exemplary sequences include, but are not limited to, those described in these latter applications and patents.

In one embodiment, the "class B" CpG oligonucleotide of the invention has the following nucleic acid sequence:

5' TCGTCGTTTTTCGGTGCTTTT 3' (SEQ ID NO: 3), or

5’ TCGTCGTTTTTCGGTCGTTTT 3’ (SEQ ID NO: 4), or

5’ TCGTCGTTTTGTCGTTTTGTCGTT 3’ (SEQ ID NO: 5), or

5’ TCGTCGTTTCGTCGTTTTGTCGTT 3’ (SEQ ID NO: 6), or

5' TCGTCGTTTTGTCGTTTTTTTCGA 3' (SEQ ID NO: 7).

In any of these sequences, all bonds may be phosphorothioate bonds. In another embodiment, in any of these sequences, one or more linkages may be phosphodiester, preferably between the “C” and “G” of the CpG motif, rendering the CpG oligonucleotide semi-flexible. In any of these sequences, ethyl-uridine or halogen can replace the 5'-T; examples of halogen substitutions include, but are not limited to, bromo-uridine or iodo-uridine substitutions.

Some non-limiting examples of B-class oligonucleotides include:

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3' (SEQ ID NO: 8), or

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3' (SEQ ID NO: 9), or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3 ' (SEQ ID NO: 10), or

5' T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3 ' (SEQ ID NO: 11), or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3 ' (SEQ ID NO: 12).

where * refers to the phosphorothioate bond.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class C CpG oligonucleotide. In one embodiment, the "class C" CpG oligonucleotides of the invention have the following nucleic acid sequences:

5' TCGCGTCGTTCGGCGCGCGCCG 3' (SEQ ID NO: 13), or

5' TCGTCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO: 14), or

5' TCGGACGTTCGGCGCGCGCCG 3' (SEQ ID NO: 15), or

5' TCGGACGTTCGGCGCGCCG 3' (SEQ ID NO: 16), or

5' TCGCGTCGTTCGGCGCGCCG 3' (SEQ ID NO: 17), or

5' TCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO: 18), or

5' TCGACGTTCGGCGCGCCG 3' (SEQ ID NO: 19), or

5' TCGCGTCGTTCGGCGCCG 3' (SEQ ID NO: 20), or

5' TCGCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO: 21), or

5' TCGTCGTTTTCGGCGCGCGCCG 3' (SEQ ID NO: 22), or

5' TCGTCGTTTTCGGCGGCCGCCG 3' (SEQ ID NO: 23), or

5' TCGTCGTTTTACGGCGCCGTGCCG 3' (SEQ ID NO: 24), or

5' TCGTCGTTTTCGGCGCGCGCCGT 3' (SEQ ID NO: 25).

In any of these sequences, all bonds may be phosphorothioate bonds. In another embodiment, in any of these sequences, one or more linkages may be phosphodiester, preferably between the “C” and “G” of the CpG motif, rendering the CpG oligonucleotide semi-flexible.

Some non-limiting examples of C-class oligonucleotides include:

5’ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 26), or

5’ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 27), or

5’ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 28), or

5’ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3’ (SEQ ID NO: 29), or

5’ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3’ (SEQ ID NO: 30), or

5’ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 31), or

5’ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3’ (SEQ ID NO: 32), or

5’ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3’ (SEQ ID NO: 33), or

5’ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 34), or

5' T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3' (SEQ ID NO: 35), or

5' T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3' (SEQ ID NO: 36), or

5' T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 3' (SEQ ID NO: 37), or

5' T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' (SEQ ID NO: 38)

where * refers to the phosphorothioate bond and _ refers to the phosphodiester bond.

In any of these sequences, ethyl-uridine or halogen can replace the 5' T; examples of halogen substitutions include, but are not limited to, bromo-uridine or iodo-uridine substitutions.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class P CpG oligonucleotide. In one embodiment, the CpG oligonucleotide for use in the present invention is a class P CpG oligonucleotide containing a 5'-TLR activation domain and at least at least two palindromic regions, one palindromic region being a 5' palindromic region of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length, either directly or by means of a spacer, where the oligonucleotide includes at least one YpR dinucleotide. In one embodiment, said oligonucleotide is not T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 27). In one embodiment, the class P CpG oligonucleotide includes at least one unmethylated CpG dinucleotide. In another embodiment, the TLR activation domain is TCG, TTTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. In yet another embodiment, the TLR activation domain is located within the 5' palindromic region. In another embodiment, the TLR activation domain is located directly 5' to the 5' palindromic region.

In one embodiment, the "class P" CpG oligonucleotides of the invention have the following nucleic acid sequence:

5' TCGTCGACGATCGGCGCGCGCCG 3' (SEQ ID NO: 39).

In these sequences, all bonds may be phosphorothioate bonds. In another embodiment, in any of these sequences, one or more linkages may be phosphodiester, preferably between the “C” and “G” of the CpG motif, rendering the CpG oligonucleotide semi-flexible. In any of these sequences, ethyl-uridine or halogen can replace the 5' T; examples of halogen substitutions include, but are not limited to, bromo-uridine or iodo-uridine substitutions.

A non-limiting example of P-class oligonucleotides includes:

5’ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3’ (SEQ ID NO: 40)

where * refers to the phosphorothioate bond and _ refers to the phosphodiester bond.

In one embodiment, the oligonucleotide includes at least one phosphorothioate linkage. In another embodiment, all internucleotide linkages of the oligonucleotide are phosphorothioate linkages. In another embodiment, the oligonucleotide includes at least one phosphodiester-like linkage. In another embodiment, the phosphodiester-like bond is a phosphodiester bond. In another embodiment, the lipophilic group is conjugated to the oligonucleotide. In one embodiment, the lipophilic group is cholesterol.

In one embodiment, all internucleotide linkages of the CpG oligonucleotides disclosed herein are phosphodiester linkages (“flexible” oligonucleotides described in PCT application WO2007/026190). In another embodiment, the CpG oligonucleotides of the invention are rendered resistant to degradation (eg, stabilized). "Stabilized oligonucleotide" refers to an oligonucleotide that is relatively resistant to degradation in vivo (eg, by exo- or endonuclease). Stabilization of the nucleic acid can be accomplished by modification of the backbone. Oligonucleotides having phosphorothioate bonds provide maximum activity and protect the oligonucleotide from degradation by intracellular exo- and endonucleases.

Immunostimulatory oligonucleotides may have a chimeric backbone that contains combinations of phosphodiester and phosphorothioate linkages. In the context of the present invention, a chimeric backbone refers to a partially stabilized backbone, wherein at least one internucleotide linkage is a phosphodiester or phosphodiester-like linkage, and where at least one other internucleotide linkage is a stabilized internucleotide linkage, wherein at least one phosphodiester or phosphodiester-like linkage and the at least one stabilized linkage are different. When the phosphodiester bond is predominantly located within a CpG motif, then such molecules are called “semi-flexible”, as described in PCT application WO2007/026190.

The size of the CpG oligonucleotide (ie, the number of nucleotide residues along the entire length of the oligonucleotide) may also contribute to the stimulatory activity of the oligonucleotide. To facilitate uptake into cells, the CpG oligonucleotide of the invention preferably has a minimum length of 6 nucleotide residues. Oligonucleotides of any size greater than 6 nucleotides (even many kb in length) are capable of inducing an immune response in the presence of sufficient immunostimulatory motifs, since larger oligonucleotides are degraded within cells. In some embodiments, the CpG oligonucleotides are from 6 to 100 nucleotides in length, preferably from 8 to 30 nucleotides. In important embodiments, the nucleic acids and oligonucleotides of the invention are not plasmids or expression vectors.

In one embodiment, the CpG oligonucleotides disclosed herein contain substitutions or modifications, for example in bases and/or sugars, as described in paragraphs 134-147 in WO2007/026190.

In one embodiment, the CpG oligonucleotide of the present invention is chemically modified. Examples of chemical modifications are known to those skilled in the art and are described, for example, in Uhlmann E. et al. (1990), Chem. Rev. 90:543, S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke, S.T. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and Hunziker J. et al., (1995), Mod. Synth. Methods 7:331–417. The oligonucleotide of the invention may have one or more modifications, where each modification is located at a specific internucleoside bridge, and/or at a specific β-D-ribose element, and/or at a specific position of the natural nucleoside base compared to an oligonucleotide with the same sequence, which consists of naturally occurring DNA or RNA.

In some embodiments of the invention, CpG-containing nucleic acids can be simply mixed with immunogenic carriers in accordance with methods known to those skilled in the art (see, for example, WO03/024480).

In a specific embodiment of the present invention, any of the immunogenic compositions disclosed herein contains from 2 μg to 100 mg CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpG oligonucleotide, preferably from 0.2 mg to 10 mg CpG -oligonucleotide, preferably 0.3 mg to 5 mg CpG oligonucleotide, even more preferably 0.5 to 2 mg CpG oligonucleotide, even more preferably 0.75 to 1.5 mg CpG oligonucleotide. In a preferred embodiment, the immunogenic composition disclosed herein contains approximately 1 mg of CpG oligonucleotide.

In a preferred embodiment, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment, the immunogenic compositions described herein contain aluminum phosphate as an adjuvant.

In preferred embodiments, the immunogenic compositions of the invention further comprise at least one component of a buffer, cryoprotectant, salt, divalent cation, nonionic detergent, free radical inhibitor, diluent or carrier.

The immunogenic composition may optionally contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include those approved by a federal, state, or other regulatory agency or listed in the United States Pharmacopoeia and other generally accepted pharmacopoeias for use in animals, including humans, as well as non-human animals. The term "carrier" can be used to refer to a diluent, adjuvant, excipient or carrier with which the pharmaceutical composition is administered. Water, saline solutions and aqueous solutions of dextrose and glycerol can be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, E. W. Martin. The composition must be appropriate for the route of administration.

The immunogenic composition may optionally contain one or more physiologically acceptable buffers selected from, but not limited to, Tris (trimethylamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In some embodiments, the composition is buffered to a pH range of from about 5.0 to about 7.0, preferably from about 5.5 to about 6.5.

The immunogenic composition may optionally contain one or more nonionic surfactants, including, but not limited to, polyoxyethylene sorbitan fatty acid esters, Polysorbate 80 (Tween 80), Polysorbate 60 (Tween 60), Polysorbate 40 (Tween 40 ) and Polysorbate-20 (Tween 20), polyoxyethylene alkyl ethers, including, but not limited to, Bridge 58, Bridge 35, as well as others such as Triton X-100; Triton X-114, NP40, Span 85 and a number of Pluronic non-ionic surfactants (eg Pluronic 121), with Polysorbate-80 components being preferred at a concentration of from about 0.001% to about 2% (up to about 0.25% being preferred ) or Polysorbate-40 at a concentration of from about 0.001% to 1% (up to about 0.5% being preferred).

The invention also relates to vaccines containing the immunogenic composition according to the invention.

Methods for inducing an immune response and protecting against infection

The present invention also includes methods of using the immunogenic compositions described in this specification. For example, in one embodiment of the invention, a method of inducing an immune response against Streptococcus pneumoniae is provided, comprising administering to a subject an immunogenic amount of any immunogenic composition described herein.

One embodiment of the invention provides a method of protecting a subject against infectionStreptococcus pneumonia, or a way to prevent infectionStreptococcus pneumonia, or a method for reducing the severity or delaying the onset of at least one symptom associated with an infection caused byStreptococcus pneumoniae,where indicated the methods involve administering to a subject an immunogenic amount of any immunogenic composition described herein.

One embodiment of the invention provides a method of protecting a subject against infectionStreptococcus pneumoniae serotype 10A, or a way to prevent infectionStreptococcus pneumoniae serotype 10A, or a method of reducing the severity or delaying the onset of at least one symptom associated with infection caused byStreptococcus pneumoniae serotype 10A,Where the methods involve administering to a subject an immunogenic amount of any immunogenic composition described herein.

In one embodiment, the invention provides a method of treating or preventing Streptococcus pneumoniae infection, disease or condition associated with Streptococcus pneumoniae serotype 10A in a subject, comprising the step of administering to the subject a therapeutically or prophylactically effective amount of an immunogenic composition described herein. In another embodiment, there is provided a method of treating or preventing Streptococcus pneumoniae infection, disease or condition associated with Streptococcus pneumoniae serotype 10A in a subject, comprising obtaining a polyclonal or monoclonal antibody preparation from an immunogenic composition described herein, and using said antibody preparation to provide the subject with passive immunity.

In one embodiment, the invention relates to the use of an immunogenic conjugate or immunogenic composition disclosed herein for the manufacture of a medicament for protecting a subject against infectionStreptococcus pneumoniae serotype 10A, and/or to prevent infectionStreptococcus pneumonia serotype 10A, and/or to reduce the severity or delay the onset of at least one symptom associated with infection caused byStreptococcus pneumoniae serotype 10A, and/or to protect the subject against infectionStreptococcus pneumoniae serotype 10A, and/or to prevent infectionStreptococcus pneumoniae serotype 10A, and/or to reduce the severity or delay the onset of at least one symptom associated with infection caused byStreptococcus pneumoniae serotype 10A.

In one embodiment of the invention, there is provided a method of protecting a subject against a Streptococcus pneumoniae serotype 22F infection , or a method of preventing a Streptococcus pneumoniae serotype 22F infection, or a method of reducing the severity or delaying the onset of at least one symptom associated with an infection caused by Streptococcus pneumoniae serotype 22F , wherein said methods involve administering to a subject an immunogenic amount of any immunogenic composition described herein.

In one embodiment, the invention provides a method of treating or preventing Streptococcus pneumoniae serotype 22F infection, disease or condition associated with Streptococcus pneumoniae serotype 22F in a subject, comprising the step of administering to the subject a therapeutically or prophylactically effective amount of an immunogenic composition described herein. In another embodiment, there is provided a method of treating or preventing Streptococcus pneumoniae serotype 22F infection, disease or condition associated with Streptococcus pneumoniae serotype 22F in a subject, comprising obtaining a polyclonal or monoclonal antibody preparation from an immunogenic composition described herein, and using said antibody preparation to provide the subject with passive immunity.

In one embodiment, the invention relates to the use of an immunogenic conjugate or immunogenic composition disclosed herein for the manufacture of a medicament for protecting a subject against infectionStreptococcus pneumoniaserotype 22F, and/or to prevent infectionStreptococcus pneumoniaserotype 22F, and/or to reduce the severity or delay the onset of at least one symptom associated with infection caused byStreptococcus pneumoniaserotype 22F, and/or to protect the subject against infectionStreptococcus pneumoniae serotype 22F, and/or to prevent infectionStreptococcus pneumoniae serotype 22F, and/or to reduce the severity or delay the onset of at least one symptom associated with infection caused byStreptococcus pneumoniae serotype 22F.

In one embodiment of the invention, there is provided a method of protecting a subject against a Streptococcus pneumoniae serotype 33F infection , or a method of preventing a Streptococcus pneumoniae serotype 33F infection, or a method of reducing the severity or delaying the onset of at least one symptom associated with an infection caused by Streptococcus pneumoniae serotype 33F , wherein said methods involve administering to a subject an immunogenic amount of any immunogenic composition described herein.

In one embodiment, the invention provides a method of treating or preventing Streptococcus pneumoniae serotype 33F infection, disease or condition associated with Streptococcus pneumoniae serotype 33F in a subject, comprising the step of administering to the subject a therapeutically or prophylactically effective amount of an immunogenic composition described herein. In another embodiment, a method is provided for treating or preventing Streptococcus pneumoniae serotype 33F infection, disease or condition associated with Streptococcus pneumoniae serotype 33F in a subject, comprising obtaining a polyclonal or monoclonal antibody preparation from an immunogenic composition described herein, and using said antibody preparation to provide the subject with passive immunity.

In one embodiment, the invention relates to the use of an immunogenic conjugate or immunogenic composition disclosed herein for the manufacture of a medicament for protecting a subject against Streptococcus pneumoniae serotype 33F infection , and/or for preventing Streptococcus pneumoniae serotype 33F infection, and/or for reducing the severity or delaying the onset of at least one symptom associated with an infection with Streptococcus pneumoniae serotype 33F , and/or to protect the subject against a Streptococcus pneumoniae serotype 33F infection and/or to prevent a Streptococcus pneumoniae serotype 33F infection, and/or to reduce the severity or delay the appearance of at least one symptom associated with infection caused by Streptococcus pneumoniae serotype 33F .

An “immune response” to an immunogenic composition is the development in a subject of a humoral and/or cellular immune response to the molecules present in the immunogenic composition or vaccine composition of interest. In the context of the present invention, a "humoral immune response" is an antibody-mediated immune response and includes the induction and formation of antibodies that recognize and bind with some affinity an antigen in an immunogenic composition or vaccine of the invention, while a "cellular immune response" is response mediated by T cells and/or other leukocytes. A “cellular immune response” is elicited through the presentation of antigenic epitopes in association with major histocompatibility complex (MHC) class I or class II molecules, CD1 or other non-classical MHC-like molecules. This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocytes ("CTLs"). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by classical or non-classical MHC and expressed on the cell surface. CTLs help induce and stimulate intracellular destruction of intracellular microbes or lysis of cells infected with such microbes. Another aspect of cellular immunity involves antigen-specific helper T cell responses. Helper T cells act to help stimulate the function and focus the activity of nonspecific effector cells against cells presenting peptide or other antigens in association with classical or nonclassical MHC molecules on their surface. "Cellular immune response" also refers to the production of cytokines, chemokines and other similar molecules produced by activated T cells and/or other leukocytes, including those derived from CD4+ and CD8+ T cells. The ability of a particular antigen or composition to stimulate a cellular immunological response can be determined by a number of assays, such as lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by analyzing antigen-specific T lymphocytes in a sensitized subject, or by measuring T-cytokine production. cells in response to repeated antigen stimulation. Such assays are well known in the art. See , for example, Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24:2369–2376.

As used herein, “treating” (including variations thereof, such as “treating” or “treated”) means any one or more of the following: (1) preventing infection or re-infection, as in the case of a traditional vaccine, (2) reducing severity or elimination of symptoms, and (3) substantial or complete elimination of the pathogen or disorder of interest. Therefore, treatment can be performed prophylactically (before infection) or therapeutically (after infection). In the present description, prophylactic treatment is the preferred method. In accordance with a specific embodiment of the present invention, compositions and methods are provided for treatment, including prophylactic and/or therapeutic immunization, of an animal host against infection with Streptococcus pneumoniae serotype 10A. In accordance with a specific embodiment of the present invention, compositions and methods are provided for treatment, including prophylactic and/or therapeutic immunization, of an animal host against infection with Streptococcus pneumoniae serotype 22F. In accordance with a specific embodiment of the present invention, compositions and methods are provided for the treatment, including prophylactic and/or therapeutic immunization, of an animal host against infection with Streptococcus pneumoniae serotype 33F. The methods of the present invention are useful for conferring prophylactic and/or therapeutic immunity on a subject. The methods of the present invention may also be practiced in biomedical research subjects.

The terms "immunogenic amount" and "immunologically effective amount", both of which are used interchangeably in this specification, refer to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either cellular (T cells) or humoral ( B cell or antibody) response, or both, measured by standard assays known to those skilled in the art.

In a preferred embodiment, said subject is a human. In the most preferred embodiment, said subject is a newborn (ie, less than three months of age), an infant (3 months to one year), or a young child (ie, one to four years of age).

In one embodiment, the immunogenic compositions disclosed herein are for use as a vaccine.

In such an embodiment, the age of the subject to be vaccinated may be less than 1 year. For example, the age of the subject to be vaccinated may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In one embodiment, the age of the subject to be vaccinated is approximately 2, 4 or 6 months. In another embodiment, the age of the subject to be vaccinated may be less than 2 years. For example, the age of the subject to be vaccinated may be approximately 12-15 months. In some cases, only one dose of the immunogenic composition of the present invention is required, but in some cases a second, third or fourth dose must be administered (see dosage regimen section).

In one embodiment of the present invention, the subject to be vaccinated is an adult 50 years of age or older, more preferably an adult 55 years of age or older. In one embodiment, the subject to be vaccinated is an adult aged 65 years or older, 70 years or older, 75 years or older, or 80 years or older.

In one embodiment, the subject to be vaccinated is an immunocompromised subject, in particular a human. An immunocompromised subject is generally defined as a person who exhibits an impaired or reduced ability to mount normal humoral or cellular defenses in response to challenge by infectious agents.

In one embodiment of the present invention, the immunocompromised subject to be vaccinated suffers from a disease or condition that impairs the immune system and produces an immune response that is insufficient to protect against or treat pneumococcal disease.

In one embodiment, said disease is a primary immunodeficiency disorder. Preferably, said primary immunodeficiency disorder is selected from the group consisting of: combined T- and B-cell immunodeficiencies, deficient antibody production, overt syndromes, diseases of immune system dysregulation, disorders of phagocytosis, congenital immunodeficiencies, autoinflammatory diseases and complement deficiency. In one embodiment, said primary immunodeficiency disorder is selected from the disorders presented on p. 24, line 11 - p. 25, line 19 of PCT application WO2010/125480.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated suffers from a disease selected from the group consisting of: HIV infection, acquired immunodeficiency syndrome (AIDS), cancer, chronic cardiac or pulmonary disorder, congestive heart failure, diabetes mellitus , chronic liver failure, alcoholism, cirrhosis of the liver, cerebrospinal fluid leak, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), splenic dysfunction (eg sickle cell anemia), lack of splenic function (asplenia), malignant blood diseases, leukemia, multiple myeloma, Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome and asthma.

In one embodiment of the present invention, the immunocompromised subject to be vaccinated is malnourished.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated takes a drug or receives treatment that reduces the body's resistance to infection. In one embodiment, said drug is selected from the drugs presented on p. 26, line 33 - p. 26, line 40 in PCT application WO2010/125480.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated is a smoker.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated has a white blood cell count (WBC) below 5 x 10 ⁹ cells per liter, or below 4 x 10 ⁹ cells per liter, or below 3 x 10 ⁹ cells per liter, or below 2 x 10 ⁹ cells per liter, or below 1 x 10 ⁹ cells per liter, or below 0.5 x 10 ⁹ cells per liter, or below 0.3 x 10 ⁹ cells per liter, or below 0.1 x 10 ⁹ cells per liter.

White blood cell count (WBC): The number of white blood cells (WBC) in the blood. WBC is usually measured as part of a CBC (complete blood count). White blood cells are infection-fighting blood cells and are different from red (oxygen-carrying) blood cells known as red blood cells. There are different types of white blood cells, including neutrophils (polymorphonuclear leukocytes; PMN), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. All types of white blood cells are reflected in the number of white blood cells in the blood. The normal range for white blood cell counts is usually between 4,300 and 10,800 cells per cubic millimeter of blood. It can also refer to the leukocyte formula and can be expressed in international units as 4.3-10.8 x 10 ⁹ cells per liter.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated suffers from neutropenia. In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated has a neutrophil count below 2 x 10 ⁹ cells per liter, or below 1 x 10 ⁹ cells per liter, or below 0.5 x 10 ⁹ cells per liter, or below 0.1 x 10 ⁹ cells per liter. liter, or below 0.05x10 ⁹ cells per liter.

Low white blood cell count or “neutropenia” is a condition characterized by abnormally low levels of neutrophils in the circulating blood. Neutrophils are a specific type of white blood cell that help prevent and fight infections. The most common reason cancer patients have neutropenia is a side effect of chemotherapy. Chemotherapy-induced neutropenia increases the patient's risk of infection and disrupts cancer treatment.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated has a CD4+ cell count below 500/mm ³ , or a CD4+ cell count below 300/mm ³ , or a CD4+ cell count below 200/mm ³ , or a CD4+ cell count below 100/mm 3 mm ³ , CD4+ cell count below 75/mm ³ , or CD4+ cell count below 50/mm ³ .

CD4 cell assays are typically reported as cell counts per mm ³ . The normal CD4 count is between 500 and 1600, and the CD8 count is between 375 and 1100. CD4 counts drop sharply in people with HIV.

In one embodiment of the invention, any immunocompromised subject disclosed herein is a male or female human.

The amount of conjugate in the composition is typically calculated based on the total polysaccharide, conjugated and unconjugated, for a given conjugate. For example, a conjugate with 20% free polysaccharide has approximately 80 μg of conjugated polysaccharide and approximately 20 μg of unconjugated polysaccharide per 100 μg polysaccharide dose. The protein contribution to the conjugate is usually not taken into account when calculating the conjugate dose. Typically, each dose contains from 0.1 to 100 μg of polysaccharide, in particular from 0.1 to 10 μg, more particularly from 1 to 10 μg, and even more particularly from 1 to 5 μg. Preferably, each dose contains about 1.1, 2, 2.2, 3, 3.3, 4, 4.4 μg of polysaccharide.

Optimal amounts of components for a particular immunogenic composition or vaccine can be established using standard studies involving observations of appropriate immune responses in subjects. After primary vaccination, subjects may receive one or more booster immunizations at appropriate intervals between them.

The effectiveness of an antigen as an immunogen can be measured either through proliferation assays, through cytolytic assays such as chromium release assays to measure the ability of T cells to lyse their specific target cells, or by measuring levels of B cell activity by measuring levels of circulating antibodies specific to to antigen, in serum. The immune response can also be determined by measuring serum levels of antigen-specific antibody induced by administration of the antigen, and more particularly by measuring the ability of antibodies thus induced to increase the opsonophagocytic ability of certain leukocytes described herein. The level of protection of the immune response can be measured by challenging the immunized host with the antigen that has been administered. For example, if the antigen to which an immune response is required is bacteria, the level of protection induced by an immunogenic amount of antigen is measured by determining the percentage of survival or percentage of mortality after challenging animals with bacterial cells. In one embodiment, the amount of protection can be measured by measuring at least one symptom associated with a bacterial infection, such as fever associated with the infection. The amount of each antigen in a multiantigen or multicomponent vaccine or immunogenic composition varies relative to each of the other components and can be determined by methods known to those skilled in the art. Such methods include procedures for measuring immunogenicity and/or potency in vivo .

The invention also provides antibodies and antibody compositions that specifically and selectively bind to the capsular polysaccharides or glycoconjugates of the present invention. In some embodiments, antibodies are produced by administering capsular polysaccharides or glycoconjugates of the present invention to a subject. In some embodiments, the invention provides purified or isolated antibodies directed against one or more capsular polysaccharides or glycoconjugates of the present invention. In some embodiments, the antibodies of the present invention are functional, as measured by bacterial killing in either an animal efficacy model or an opsonophagocytic killing assay. In some embodiments, antibodies of the invention confer passive immunity on a subject. The present invention provides polynucleotide molecules encoding an antibody or antibody fragment of the invention, and cells, cell lines (such as hybridoma cells or other engineered cell lines for recombinant antibody production), or transgenic animals that produce an antibody or antibody composition of the invention, using methods well known to those skilled in the art.

Examples

Example 1: Preparation of serotype 22F-CRM polysaccharide conjugate ₁₉₇

1.1. Preparation of isolated polysaccharide of Streptococcus pneumoniae serotype 22F

Serotype 22F capsular polysaccharides can be obtained directly from bacteria using isolation methods known to those skilled in the art (see, for example, the methods described in US Patent Application Publications 20060228380, 20060228381, 20070184071, 20070184072, 20070231340 and 200801 02498 or in WO2008118752). Streptococcus pneumonia serotype 22F was grown in a seed bottle and then transferred to a seed fermenter. When the required optical density was reached, the cells were transferred to a production fermenter. The fermentation broth was inactivated by the addition of N-lauroylsarcosine and purified by ultrafiltration and diafiltration.

Purified polysaccharideStreptococcus pneumoniaeserotype 22F was brought to the desired size by high pressure homogenization using a PANDA 2K homogenizer® (GEA Niro Soavi) to obtain the isolated polysaccharideStreptococcus pneumoniaeserotype 22F.

1.2. Oxidation of isolated capsular polysaccharide of Streptococcus pneumoniae serotype 22F

Oxidation of the polysaccharide was carried out in a 100 mM potassium phosphate buffer (pH 5.8 ± 0.2), obtained by sequentially adding a calculated amount of 500 mM potassium phosphate buffer (pH 5.8) and WFI (water for injection) to obtain the final polysaccharide concentration 2.0 g/l. If necessary, the pH of the reaction mixture was adjusted to about 5.8. After pH adjustment, the reaction temperature was reduced to 5±3°C. Oxidation was initiated by adding 0.10 ± 0.02 molar equivalents (m-eq) of sodium periodate. The target oxidation reaction time is 16±1 h at 5±3°C.

The oxidation reaction was quenched with 2 mEq of 2,3-butanediol with continuous stirring at 5±3°C for 1-2 hours.

Concentration and diafiltration of the activated polysaccharide was performed using 100K MWCO (molecular weight cutoff) ultrafiltration cassettes. Diafiltration was performed against 35x WFI diavolume. The purified activated polysaccharide was stored at 5±3°C. The purified activated saccharide is characterized, among other things, by (1) molecular weight determined by SEC-MALLS (2) presence of O-acetyl and (3) oxidation state.

SEC-MALLS are used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate polysaccharides by hydrodynamic volume. Refractive index (RI) detectors and multi-angle laser light scattering (MALLS) detectors are used to determine molecular weight. When light interacts with a substance, it is scattered, and the amount of light scattered is related to the concentration, the square of dn/dc (specific increments of the refractive index), and the molar mass of the substance. The molecular weight measurement is calculated from the scattered light signal from the MALLS detector and the concentration signal from the RI detector.

The degree of oxidation (DO = moles of sugar repeat unit/moles of aldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit are determined by various colorimetric methods, such as, for example, the anthrone method. The polysaccharide is first broken down into monosaccharides by sulfuric acid and heat. The anthrone reagent reacts with hexoses to form a yellow-green complex, the absorbance of which is read spectrophotometrically at 625 nm. In the assay, uptake is directly proportional to the amount of hexose present.

Aldehyde moles are also determined simultaneously using the MBTH colorimetric method. The MBTH assay involves the formation of an azine compound by reacting aldehyde groups (from this sample) with 3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). Excess 3-methyl-2-benzothiazoline hydrazone is oxidized to form a reactive cation. The reactive cation and the azide react to form a blue chromophore. The resulting chromophore is then read spectroscopically at 650 nm.

1.3. Conjugation of activated polysaccharide of Streptococcus pneumoniae serotype 22F with CRM ₁₉₇

The conjugation method consists of the following stages:

a. Mixing with sucrose excipient and lyophilization.

b. Recovery of lyophilized polysaccharide and CRM ₁₉₇ .

V. Conjugation of activated polysaccharide to CRM ₁₉₇ and blocking.

d. Purification of the conjugate.

a) Mixing with sucrose and lyophilization

The activated polysaccharide was mixed with sucrose (50% w/v in WFI) at a ratio of 25 g sucrose per gram activated polysaccharide. The bottle of the mixture was then lyophilized. After lyophilization, the bottles containing the lyophilized activated polysaccharide were stored at
-20±5°C. The calculated amount of CRM ₁₉₇ protein (target input S/P ratio = 1) was superficially frozen and lyophilized separately. Lyophilized CRM ₁₉₇ was stored at -20±5°C.

b) Recovery of lyophilized activated polysaccharide and protein CRM ₁₉₇

The lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). After complete dissolution of the polysaccharide, an equal amount of anhydrous DMSO was added to the lyophilized CRM ₁₉₇ for reconstitution.

c) Conjugation of the activated polysaccharide with CRM ₁₉₇ and blocking

Dissolved CRM ₁₉₇ (in DMSO) was combined in a vessel for conjugation with the reduced activated polysaccharide. The final concentration of polysaccharide in the reaction solution was 1 g/L. Conjugation was initiated by adding 1.5±0.1 mEq sodium cyanoborohydride to the reaction mixture and the reaction mixture was incubated at 23±2°C for 20±2 hours. The conjugation reaction was terminated by adding 2 mEq sodium borohydride. The blocking reaction mixture was incubated at 23 ± 2°C for 3 ± 1 h.

d) Purification of the conjugate

The conjugate solution was diluted 1:5 with chilled 5 mM succinate-0.9% sodium chloride solution (pH 6.0) in preparation for purification by tangential flow filtration using 100K MWCO membranes and 20X diafiltration was performed using 5 mM succinate-0 solution .9% sodium chloride solution (pH 6.0) as medium. After completion of diafiltration, the retentate conjugate was further diluted, filtered through a 0.22 μm filter and stored at 2-8°C.

Table 1 presents the characteristic data for polysaccharide serotype 22F-CRM ₁₉₇ conjugates obtained by the method of the invention. In particular, conjugates 5 and 6 from the table were obtained as described in example 1.

Serotype 22F-CRM ₁₉₇ polysaccharide conjugates produced by the RAC-DMSO process provide superior yields and exhibit better batch-to-batch MW (molecular weight) reproducibility along with significantly lower % free saccharide levels and higher carrier protein modification ( lysine) compared to conjugates obtained by the RAC-aqueous method, as shown in Table 1.

Table 1 - Conjugates of serotype 22F Conjugate No 1 2 3 4 5 6 Water Water Water DMSO DMSO DMSO Act. polysaccharide
MW, kDa 488 803 410 639 709 416 DO 17.3 15 10.3 14.6 14.4 13.7 Input ratio 0.8 0.8 1 0.8 1 1 % yield of conjugate 66 63 46 70 62 75 % free saccharide 18 43 45 < 5 < 5 8 MW conjugate 1024 3544 462 3911 3734 4453 Saccharide-protein ratio 1.6 1.1 1.45 0.8 0.65 1.0 modified Lys (AAA) 2.3 N/A N/A 6.1 N/A 4.7

The % conjugate yield is calculated as follows: (amount of polysaccharide in conjugate x100)/amount of activated polysaccharide.

Example 2: Preparation of serotype 10A-CRM polysaccharide conjugate ₁₉₇

2.1. Preparation of isolated polysaccharide of Streptococcus pneumoniae serotype 10A

The isolated serotype 10A polysaccharide was prepared as described in Example 1.1, except that the purified polysaccharide was not brought to the desired size.

2.2. Oxidation of isolated capsular polysaccharide of Streptococcus pneumoniae serotype 10A

A calculated volume of 0.1 M potassium phosphate buffer (pH 6.0) and water for injection (WFI) was added to the polysaccharide solution to give a final polysaccharide concentration of 2.5 g/L and a final potassium phosphate buffer concentration of 25 mM, if necessary. The pH was adjusted to approximately 6.0. The diluted polysaccharide was then cooled to 5±3°C. Oxidation was initiated by adding 0.25 ± 0.02 molar equivalents (mEq) of sodium periodate solution. The oxidation reaction time was approximately 4 hours at 5±3°C. The oxidation reaction was quenched with 1 mEq of 2,3-butanediol with continuous stirring at 5±3°C for 1-2 hours.

After reaching the target reaction time, the activated polysaccharide was concentrated using 30K MWCO Millipore ultrafiltration cassettes. Diafiltration was then performed against 20x WFI diavolume. The purified activated polysaccharide was stored at 5±3°C. The purified activated saccharide is characterized, among other things, by (1) molecular weight determined by SEC-MALLS and (2) oxidation state.

2.3 Conjugation of activated polysaccharide of Streptococcus pneumoniae serotype 10A with CRM ₁₉₇

The conjugation method consists of the following stages:

a. Mixing with sucrose excipient and lyophilization.

b. Recovery of lyophilized polysaccharide and CRM197.

V. Conjugation of activated polysaccharide to CRM197 and blocking

d. Purification of the conjugate

a) Mixing with sucrose

The activated polysaccharide was mixed with sucrose in a ratio of 25 ± 2.5 grams of sucrose per gram of activated polysaccharide. The bottle of the mixture was then lyophilized. After lyophilization, the bottles containing the lyophilized activated polysaccharide were stored at -20±5°C.

b) Recovery of lyophilized activated polysaccharide and CRM ₁₉₇ protein.

The lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). Once the polysaccharide was completely dissolved, the same amount of anhydrous DMSO was added to the calculated CRM ₁₉₇ for reduction.

c) Conjugation of the activated polysaccharide with CRM ₁₉₇ and blocking

Reduced CRM ₁₉₇ (in DMSO) was added to the reduced activated polysaccharide in the conjugation reactor. The final concentration of the polysaccharide was 1 g/L. Conjugation was performed by adding 1.2 ± 0.1 mEq of sodium cyanoborohydride to the reaction mixture. The reaction mixture was incubated at 23±2°C for 24±2 hours. The conjugation reaction was stopped by adding 2 mEq of sodium borohydride. The blocking reaction mixture was incubated at 23 ± 2°C for 3 ± 1 h.

The conjugation reaction was stopped by adding 2 mEq of sodium borohydride. This blocking reaction continued for 3±1 hours at 23±2°C.

d) Purification of the conjugate

The conjugate solution was then diluted in 5x (v/v) chilled 5 mM succinate-0.9% sodium chloride solution (pH 6.0 and 20X diafiltration was performed using 5 mM succinate-0.9% sodium chloride solution (pH 6.0 After completion of the initial diafiltration, the conjugate retentate was passed through a 0.22 µm filter.The conjugate was further diluted with 5 mM succinate/0.9% sodium chloride solution (pH 6) and, after a final filtration step through a 0.22 µm filter, was stored at 2-8°C.

Table 2 presents data for serotype 10A-CRM ₁₉₇ polysaccharide conjugates obtained by the method of the invention. Specifically, conjugates 4-6 in Table 2 were prepared as described in Example 2.

Serotype 10A-CRM ₁₉₇ polysaccharide conjugates produced by the RAC-DMSO process provide superior yields and exhibit better batch-to-batch MW reproducibility along with significantly lower % free saccharide levels and higher levels of carrier protein (lysine) modification. compared to conjugates obtained by the RAC-aqueous method, as shown in Table 2.

Table 2. Serotype 10A conjugates Conjugate No 1 2 3 4 5 6 Water Water Water DMSO DMSO DMSO DO 12.2 12.2 12.8 10.3 10.8 10.5 MW of activated saccharide, kDa 157 157 163 170 170 170 Input ratio 1.0 1.0 1.0 1.1 1.1 1.1 % yield 71 34 50 82 73 66 % free saccharide 20 31 26 6.8 6.4 9.7 MW of conjugate, kDa 1550 968 3050 4034 3463 5540 Saccharide-protein ratio 1.6 2.6 1.6 1.1 1.2 1.0 Lys modification AAA 2.6 N/A N/A 6.9 6.7 6.1

Example 3: Preparation of serotype 33F-CRM polysaccharide conjugate ₁₉₇

3.1. Preparation of isolated polysaccharide of Streptococcus pneumoniae serotype 33F

The isolated polysaccharide serotype 33F was prepared as described in example 1.1.

3.2. Oxidation of isolated capsular polysaccharide of Streptococcus pneumoniae serotype 33F

A calculated volume of 0.1 M sodium phosphate buffer (pH 6.0) and water for injection (WFI) was added to the polysaccharide solution to obtain a final polysaccharide concentration of 2 g/L. If necessary, the pH was adjusted to approximately 6.0. The diluted polysaccharide was then cooled to 5±3°C. Oxidation was initiated by adding 0.1 molar equivalent (mEq) sodium periodate solution. The oxidation reaction time was approximately 20 hours at 5±3°C. The oxidation reaction was quenched with 1 mEq of 2,3-butanediol with continuous stirring at 5±3°C for approximately 1 hour. After reaching the target reaction time, the activated polysaccharide was concentrated using 100K MWCO Millipore ultrafiltration cassettes. Diafiltration was then performed against 40x WFI diavolume. The purified activated polysaccharide was stored at 5±3°C. The purified activated saccharide is characterized, among other things, by (1) molecular weight according to SEC-MALLS and (2) oxidation state.

3.3 Conjugation of activated polysaccharide of Streptococcus pneumoniae serotype 33F to CRM ₁₉₇

The serotype 33F-CRM197 polysaccharide conjugate was prepared in a manner similar to that of Example 1.3, using the activated 33F polysaccharide prepared in Example 3.2.

Serotype 33F-CRM ₁₉₇ polysaccharide conjugates were prepared via the RAC-DMSO process, providing better yields and demonstrating better batch-to-batch reproducibility in maintaining O-acetyl levels along with lower % free saccharide levels and higher levels of carrier protein modification (lysine) compared to conjugates obtained by the RAC-aqueous method, as shown in Table 3.

Table 3 - Conjugates of serotype 33F Conjugate No 1 2 3 4 5 6 Vodn. Vodn. Vodn. DMSO DMSO DMSO DO 15.2 15.5 15.7 17 9 14 MW activated polysaccharides 1126 573 574 120 328 128 % yield 65 51 48 78 86 66 % free saccharides 8.5 19 13 18 5 < 5 MW of conjugate, kDa 1341 2137 3674 3158 2165 2001 Saccharide-protein ratio 1.7 1.4 1.3 1.1 1.1 0.79 % O-Ac Saved <LOQ <LOQ <LOQ 82 100% 100% Conjug. Lys (AAA) N/A 2.3 2.3 4.5 3.4 5.2

LOQ = Limit of Quantification

Example 4: Effect of Quenching on the Molecular Weight of Activated Polysaccharides Serotypes 22F and 33F

Activation of polysaccharides 22F and 33F was performed at 4°C or 22°C. Both temperatures provided similar DO values. However, due to the fact that the increased temperature resulted in a faster destruction of the activated polysaccharide, thus reducing the MW of the activated polysaccharide (as shown, for example, in Fig. 4 (22°C) and Fig. 5 (4°C)) , a temperature of 4°C is generally preferred for the activation reaction. In addition, activation data showed that quenching of unreacted NaIO ₄ after oxidation is an important activation step, especially activation using more NaIO ₄ . The graph shown in Fig. 6 shows that the MW of activated polysaccharide 33F is relatively stable when increased concentrations of periodate are used for activation (with quenching), while such MW is highly variable when no quenching step is used. Under certain conditions established to obtain the target oxidation state, the molecular weight of the activated polysaccharide is less variable when using a quenching step. The addition of a quencher after oxidation helps maintain the structural integrity of the activated polysaccharide until purification is completed, such as through ultrafiltration and diafiltration.

Example 5: Opsonophagocytic Activity Assay (OPA)

The immunogenicity of conjugates produced by the methods disclosed herein can be assessed using the opsonophagocytic assay (OPA) described below.

Groups of thirty 6-7 week old female Swiss Webster mice were immunized with 0.001 μg, 0.01 μg, or 0.1 μg of test conjugates via subcutaneous administration at week 0. Mice were further administered the same dose of conjugate at week 3 and then bled at week 4. Serotype-specific OPAs were performed on 4-week serum samples.

Validated opsonophagocytic activity (OPA) assays are used to measure functional antibodies in mouse serum specific to S. pneumonia serotypes 10A, 22F, or 33F. The test serum is placed in assay reaction mixtures that measure the ability of capsular polysaccharide-specific immunoglobulin to opsonize bacteria, trigger complement precipitation, thereby promoting phagocytosis and killing of bacteria by phagocytes. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial counts compared to control wells without test serum. The OPA titer is interpolated from two dilutions that include this 50% kill limit.

OPA procedures were based on the methods described in Hu et al., Clin Diagn Lab Immunol 2005;12(February (2)):287-95 with the following modifications. The test serum was serially diluted 2.5-fold and added to microtiter plates. Live target bacterial strains serotypes 10A, 22F and 33F were added to the wells and the plates were shaken at 25°C (serotype 22F) or 37°C (serotype 10A and 33F) for 30 minutes. Serum from differentiated HL-60 cells (phagocytes) and baby rabbits (3 to 4 weeks of age, Pel-Freez®, 12.5% final concentration) was added to the wells and the plates were shaken at 37°C for 45 minutes (serotype 22F and 33F) or 60 minutes (serotype 10A). To terminate the reaction, 80 μl of 0.9% NaCl was added to all wells, mixed, and a 10 μl aliquot was transferred to the wells of MultiScreen HTS HV filter plates (Millipore®) containing 200 μl of water. The liquid was filtered through the plates under vacuum and 150 µl of HySoy medium was added to each well and filtered through it. Filter plates were then incubated at 37°C, 5% CO ₂ overnight and then fixed with Destain Solution (Bio-Rad). The plates were then stained with Coomassie blue and destained once. Colonies were visualized and counted on a Cellular Technology Limited (CTL) ImmunoSpot Analyzer®. Counts of untreated colonies were used to construct death curves and determine OPA titers.

Serotype 10A-CRM ₁₉₇ polysaccharide conjugates, serotype 22F-CRM ₁₉₇ polysaccharide conjugates, and serotype 33F-CRM ₁₉₇ polysaccharide conjugates prepared as described in Examples 1-3 were tested in the OPA assay described above and were found to be immunogenic.

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List of sequences

<110> Pfizer Inc.

<120> Capsular polysaccharides of Streptococcus pneumoniae and their

conjugates

<130>PC71942A

<150> US 61/929,598

<151> 2014-01-21

<160> 40

<170> Patent In version 3.5

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ggggacgacg tcgtgggggg g

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tcgtcgtttt tcggtgcttt t

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tcgtcgtttt tcggtcgttt t

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tcgtcgtttc gtcgttttgt cgtt

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tcgtcgtttt gtcgtttttt tcga

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tcgcgtcgtt cggcgcgcgc cg

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tcgtcgacgt tcggcgcgcg ccg

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tcggacgttc ggcgcgcgcc g

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tcggacgttc ggcgcgccg

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tcgcgtcgtt cggcgcgccg

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tcgacgttcg gcgcgcgccg

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tcgacgttcg gcgcgccg

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tcgcgtcgtt cggcgccg

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tcgcgacgtt cggcgcgcgc cg

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tcgtcgtttt cggcgcgcgc cg

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tcgtcgtttt cggcggccgc cg

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tcgtcgtttt acggcgccgt gccg

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tcgtcgtttt cggcgcgcgc cgt

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tcgcgtcgtt cggcgcgcgc cg

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tcgtcgacgt tcggcgcgcg ccg

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tcggacgttc ggcgcgcgcc g

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tcgacgttcg gcgcgcgccg

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tcgacgttcg gcgcgccg

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tcgcgtcgtt cggcgccg

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tcgcgacgtt cggcgcgcgc cg

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tcgtcgtttt cggcgcgcgc cg

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tcgtcgtttt cggcggccgc cg

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tcgtcgtttt acggcgccgt gccg

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tcgtcgtttt cggcgcgcgc cgt

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tcgtcgacga tcggcgcgcg ccg

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<---

Claims (33)

1. An immunogenic conjugate suitable for the manufacture of a medicament for protecting a subject against Streptococcus pneumoniae infection, comprising a polysaccharide of Streptococcus pneumoniae serotype 10A, 22F or 33F covalently linked to a carrier protein, prepared by a method comprising the following steps: (a) reacting an isolated capsular polysaccharide of serotype 10A, 22F or 33F with 0.05-0.2 molar periodate equivalents for 22F, 0.1-0.3 molar periodate equivalents for 10A, or 0.05-0.2 molar periodate equivalents for 33F; (b) quenching the oxidation reaction by adding a quenching agent in an amount of 1-3 molar equivalents for 22F, 0.5-2 molar equivalents for 10A, 0.5-2 molar equivalents for 33F to obtain an activated polysaccharide of serotype 10A, 22F or 33F, wherein the oxidation state of activated serotype 10A is from 8 to 14, activated serotype 22F is from 10 to 20, and activated serotype 33F is from 9 to 18; (c) mixing said activated polysaccharide of serotype 10A, 22F or 33F with a carrier protein; And (d) interaction of a mixture of an activated polysaccharide of serotype 10A, 22F or 33F and a carrier protein with a reducing agent to form a polysaccharide of serotype 10A, 22F or 33F: carrier protein conjugate, where the degree of conjugation of said immunogenic conjugate is from 2 to 15. 2. Immunogenic conjugate according to claim 1, where said method additionally includes the step of: (e) blocking unreacted aldehydes in the serotype 10A, 22F or 33F polysaccharide: carrier protein conjugate. 3. The immunogenic conjugate according to claim 1 or 2, where steps (a) and (b) are performed in DMSO (dimethyl sulfoxide) or in an aqueous solution. 4. Immunogenic conjugate according to any one of paragraphs. 1-3, where the initial ratio of activated polysaccharide of serotype 10A, 22F or 33F to the carrier protein at stage (c) is from 5:1 to 0.1:1, from 2:1 to 0.1:1, from 2: 1 to 1:1, 1.5:1 to 1:1, 0.1:1 to 1:1, 0.3:1 to 1:1 or 0.6:1 to 1.2: 1. 5. Immunogenic conjugate according to any one of paragraphs. 1-4, where in step (d) the activated polysaccharide reacts with 0.5-2.5 molar equivalents of a reducing agent. 6. Immunogenic conjugate according to claim 5, where the reducing agent is sodium cyanoborohydride. 7. Immunogenic conjugate according to any one of paragraphs. 1-6, where the oxidizing agent is sodium metaperiodate. 8. Immunogenic conjugate according to any one of paragraphs. 1-7, where the reaction temperature in steps (a) and (b) is maintained from 1 to 10°C. 9. Immunogenic conjugate according to any one of paragraphs. 1-8, wherein the quenching agent is selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid. 10. Immunogenic conjugate according to any one of paragraphs. 1-8, wherein the quenching agent is a compound of the formula (II), wherein each of R ¹ and R ² is independently selected from H, methyl, ethyl, propyl or isopropyl. 11. Immunogenic conjugate according to any one of paragraphs. 1-8, wherein the quenching agent is selected from glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol. 12. Immunogenic conjugate according to any one of paragraphs. 1-8, where the quenching agent is butane-2,3-diol. 13. Immunogenic conjugate according to any one of paragraphs. 1-12, which has a molecular weight from 500 to 15000; from 500 to 10000; from 2000 to 10000; from 3000 to 8000 kDa or from 3000 to 5000 kDa. 14. Immunogenic conjugate according to any one of paragraphs. 1-13, wherein the ratio of serotype 10A, 22F or 33F polysaccharide to carrier protein in the conjugate is from 0.4 to 2.0. 15. Immunogenic conjugate according to any one of paragraphs. 1-14, where the degree of conjugation of the specified immunogenic conjugate is from 2 to 13, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 5, from 2 to 4, from 3 to 15, from 3 to 13 , from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 5 to 15, from 5 to 10, from 8 to 15, from 8 to 12, from 10 to 15 or from 10 to 12. 16. Immunogenic conjugate according to any one of paragraphs. 1-15, wherein said conjugate is a 22F conjugate and wherein said conjugate contains at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 22F polysaccharide. 17. Immunogenic conjugate according to any one of paragraphs. 1-16, wherein said conjugate is a 22F conjugate and wherein the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the isolated polysaccharide is at least 0.7. 18. Immunogenic conjugate according to any one of paragraphs. 1-17, wherein said conjugate is a 22F conjugate and wherein the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the activated polysaccharide is at least 0.7. 19. Immunogenic conjugate according to any one of paragraphs. 1-15, wherein said conjugate is a 33F conjugate and wherein said conjugate contains at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 33F polysaccharide. 20. Immunogenic conjugate according to any one of paragraphs. 1-15, wherein said conjugate is a 33F conjugate and wherein the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the isolated polysaccharide is at least 0.7. 21. Immunogenic conjugate according to any one of paragraphs. 1-15, wherein said conjugate is a 33F conjugate and wherein the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the activated polysaccharide is at least 0.7. 22. Immunogenic conjugate according to any one of paragraphs. 1-21, where the carrier protein is CRM197. 23. An immunogenic composition suitable for the manufacture of a medicinal product for protecting a subject against infection by Streptococcus pneumoniae , containing an immunogenic conjugate according to any one of paragraphs. 1-22 and a physiologically acceptable carrier. 24. The immunogenic composition according to claim 23, further containing at least one additional antigen. 25. Immunogenic composition according to claim 23 or 24, additionally containing an adjuvant. 26. Immunogenic composition according to claim 25, where the adjuvant is aluminum phosphate. 27. A vaccine suitable for protecting a subject against infection with Streptococcus pneumoniae , containing an immunogenic composition according to any one of paragraphs. 23-26.