COMPOSITION OF THE MULTIVALENT PNEUMOCOCCAL PROTEIN-POLYSACCHARIDE CONJUGATE

Abstract

Multivalent pneumococcal conjugate compositions with mixed carriers are provided, comprising 21 different pneumococcal capsular protein-polysaccharide conjugates, wherein each of the conjugates includes a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated with tetanus toxoid (TT) or CRM 197 , wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, wherein the capsular polysaccharides of two of the serotypes 1, 3, and 5 and one or both of the two serotypes 15B and 22F are conjugated with TT and the remaining capsular polysaccharides are conjugated with CRM 197 Methods for producing mixed-carrier multivalent pneumococcal conjugate compositions and methods for using these for prophylaxis against Streptococcus pneumoniae infection or disease in a subject are also provided.

Description

IMMUNOGENIC CONJUGATES OF STREPTOCOCCUS PNEUMONIAE SEROTYPE 9N AND VACCINE COMPOSITION COMPRISING THE SAME TECHNICAL FIELD This description relates to a Streptococcus pneumoniae vaccine, more particularly to an immunogenic conjugate of Streptococcus pneumoniae serotype 9N and a vaccine composition containing the same. BACKGROUND OF ART Streptococcus pneumoniae is a major cause of pneumonia. It also causes invasive diseases such as sepsis, bacteremia, meningitis, and others. According to Statistics Korea [Statistics on Causes of Death in Korea, 2014], pneumonia mortality in 2014 increased by 11% annually to 23.7 deaths per 100,000 people. In 2015, it increased 2.8 times. As such, pneumonia mortality is steadily rising. Furthermore, according to a 2012 WHO report, 476,000 HIV-negative infants and children aged 5 and under died from Streptococcus pneumoniae infection in 2008 worldwide. This represents 5% of all deaths of infants and children aged 5 and under. To prevent diseases caused by Streptococcus pneumoniae, in 1977, Dr. Robert Austrian developed a 14-valent polysaccharide vaccine, which was later developed into a 23-valent polysaccharide vaccine. Multivalent pneumococcal polysaccharide vaccines have proven useful in preventing Streptococcus pneumoniae diseases in elderly and high-risk patients. However, most infants and children do not IF-2019-79554467-APN-ANP#INPI Page 1 of 67 shows an immune response to the Streptococcus pneumoniae polysaccharide, because the immune response is independent of T cells. Therefore, a conjugate vaccine of Streptococcus pneumoniae capsular polysaccharide and carrier protein was developed, capable of inducing a T cell-dependent response. The heptavalent Streptococcus pneumoniae conjugate vaccine (Prevnar®) contains capsular polysaccharides derived from the 7 most prevalent serotypes: 4, 6B, 9V, 14, 18C, 19F, and 23F. Later, the 13-valent conjugate vaccine Prevnar 13®, which contains an additional 6 serotypes (1, 3, 5, 6A, 7F, and 19A), and the 10-valent conjugate vaccine Synflorix, which contains an additional 3 serotypes (1, 5, and 7F), were successively developed, and the number of invasive diseases caused by Streptococcus pneumoniae also decreased. However, due to the replacement of the serotype with the introduction of Prevnar, Prevnar 13 and Synflorix and the reduction in the number of diseases due to the serotypes contained in the vaccines, the importance of non-vaccine serotypes, which have been considered less important, is emphasized. In particular, although serotype 9N was not included in the existing vaccine, a recent study revealed that serotype 9N exhibits higher mortality than the frequently encountered serotype 14. According to the serotype distribution of IPD cases investigated in EU / EEA countries in 2010, serotype 9N was one of the leading causes of IPD, ranking 13th. Furthermore, there is the problem that general saccharin vaccines do not provide continuous immunization against serotype 9N. Despite the increased incidence and risk of pneumococcal diseases caused by Streptococcus pneumoniae serotype 9N, studies IF-2019-79554467-APN-ANP#INPI Page 2 of 67 on effective prevention or treatment of serotype 9N infection are insufficient. Therefore, there is a growing need for an immunogenic conjugate and immunogenic composition containing the non-vaccine serotype, which has been included in a 23-valent saccharide vaccine to provide broader protection but not in a conjugate vaccine. DESCRIPTION Technical Problem This description is intended to provide a multivalent vaccine capable of providing broad protection. This description is also intended to provide a pneumococcal conjugate vaccine that has a higher antibody titer. The inventors intend to provide excellent immunogenicity against pneumococcal infection caused by serotype 9N. Technical solution The present description provides an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, containing: a serotype 9N capsular saccharide derived from Streptococcus pneumoniae; and a carrier protein bound to the capsular saccharide. In an exemplary embodiment of the present description, the saccharide of serotype 9N can bind to the carrier protein to form a conjugate in a state in which it is activated and has a molecular weight of 200-700 kDa. The immunogenic conjugate may have a molecular weight of 500-4,000 kDa and the carrier protein may be CRMi97. IF-2019-79554467-APN-ANP#INPI Page 3 of 67 A (P / P) ratio of the capsular saccharide of serotype 9N with respect to the carrier protein in the immunogenic conjugate can be 0.1-5, specifically 0.5-2.5. In an exemplary embodiment, 15-60% of the immunogenic conjugate can have a Kd of 0.3 or lower on a CL-4B column. In one exemplary embodiment, the conjugate can have an oxidation degree of 2-19. In an exemplary embodiment of the present description, when the saccharide from Streptococcus pneumoniae serotype 9N is conjugated to the protein by adding 0.02-0.19 μg of periodate per 1 μg of sugar, the conjugate can have a molecular weight of 500-4,000 kDa, a molecular weight distribution of 15-60% (Kd < 0.3) and a saccharide / protein ratio of 0.5-2.5. The present description also provides an immunogenic composition containing the immunogenic conjugate and a physiologically acceptable vehicle. This description also provides a vaccine containing the immunogenic composition. This description also provides a method for preparing an immunogenic conjugate of Streptococcus pneumoniae serotype 9N, which includes: (a) a step of lysing a bacterial cell that produces the capsular polysaccharide of Streptococcus pneumoniae serotype 9N by fermentation; (b) a step for purifying the capsular saccharide of Streptococcus pneumoniae serotype 9N from the lysed cell; IF-2019-79554467-APN-ANP#INPI Page 4 of 67 (c) an activation step of the saccharide by reaction with an oxidizing agent; and (d) a step of forming a conjugate of the capsular saccharide of Streptococcus pneumoniae serotype 9N bound to a carrier protein by mixing the activated saccharide with the carrier protein. The carrier protein mixed in step (d) can be reacted with a reducing agent to form the conjugate with the activated saccharide. In step (c), 0.02-0.19 μg of periodate can be reacted with 1 μg of saccharide at 20-25°C for 15-20 hours. The saccharide that reacted with the oxidizing agent in step (c) can have a molecular weight of 400-900 kDa. The activated saccharide mixed with the carrier protein in step (d) can have a molecular weight of 200-700 kDa. The immunogenic conjugate can have a molecular weight of 500-4,000 kDa. An initial input ratio of carrier protein to activated 9N serotype capsular saccharide (carrier protein: saccharide) can be 0.5-2.5:1. In an exemplary embodiment, at least 15-60% of the immunogenic conjugate can have a Kd of 0.3 or lower on a CL-4B column. This description also provides an immunogenic conjugate obtained by the preparation method described above. This description also provides an immunogenic composition containing an immunogenic conjugate obtained by the preparation method and a physiologically acceptable vehicle. This description also provides a vaccine containing the immunogenic composition. IF-2019-79554467-APN-ANP#INPI Page 5 of 67 Specifically, when the Streptococcus pneumoniae serotype 9N polysaccharide of the present description is conjugated to the protein by the addition of 0.02-0.19 μg of periodate per 1 μg of sugar, the conjugate can have a molecular weight of 500-4,000 kDa, a molecular weight distribution of 15-60% (Kd < 0.3) and a polysaccharide / protein ratio of 0.5-2.5. Definition of terms The term saccharide or capsular saccharide used in this description refers to a capsular saccharide, polysaccharide, or oligosaccharide derived from Streptococcus pneumoniae serotypes. The term immunogenic conjugate or conjugate used herein refers to a capsular saccharide, polysaccharide, or oligosaccharide covalently conjugated to a carrier protein. It may also be called a capsular saccharide-protein conjugate. The term activated saccharide (polysaccharide) used herein is understood to refer to an oxidized capsular polysaccharide and may be understood to refer to a polysaccharide in a state in which the capsular polysaccharide has not yet been conjugated to a carrier protein. The oxidized capsular polysaccharide may further be lyophilized and, in this case, may be understood to refer to a polysaccharide in a state in which it has not yet been conjugated to a protein after lyophilization. The activated polysaccharide described herein may be hydrolyzed before activation. As used in this document, a percentage concentration is a weight / volume (w / v) concentration or a weight / weight (w / w) concentration. IF-2019-79554467-APN-ANP#INPI Page 6 of 67 Unless otherwise specified, the molecular weight of a capsular saccharide or a capsular saccharide-carrier protein conjugate refers to the average molecular weight calculated by size exclusion chromatography (SEC) in combination with multi-angle laser light scattering (MALLS). The term degree of oxidation (DO) refers to the number of sugar repeating units per aldehyde group generated when a purified or sized saccharide is activated with an oxidizing agent. The degree of oxidation of a saccharide can be determined using routine methods known to those skilled in the art. Detailed description 1. Immunogenic composition. The immunogenic composition of the present description may contain an immunogenic conjugate containing a capsular polysaccharide or antigen oligosaccharide covalently conjugated to a carrier, specifically a carrier protein. The capsular oligosaccharide or polysaccharide antigen of the immunogenic conjugate is derived from the serotype(s) of Streptococcus pneumoniae. Specifically, Streptococcus pneumoniae can have at least 24 different serotypes (ov, valencies), including serotype 9N. That is, it can have 24 capsular saccharides. In an exemplary form of realization, there are 2 different serotypes. In an exemplary form of realization, there are 3 different serotypes. In an exemplary form of realization, there are 4 different serotypes. In an exemplary form of realization, there are 5 different serotypes. In an exemplary form of realization, IF-2019-79554467-APN-ANP#INPI Page 7 of 67. There are 6 different serotypes. In an exemplary embodiment, there are 7 different serotypes. In an exemplary embodiment, there are 8 different serotypes. In an exemplary embodiment, there are 9 different serotypes. In an exemplary embodiment, there are 10 different serotypes. In an exemplary embodiment, there are 11 different serotypes. In an exemplary embodiment, there are 12 different serotypes. In an exemplary embodiment, there are 13 different serotypes. In an exemplary embodiment, there are 14 different serotypes. In an exemplary embodiment, there are 15 different serotypes. In an exemplary embodiment, there are 16 different serotypes. In an exemplary embodiment, there are 17 different serotypes. In an exemplary embodiment, there are 18 different serotypes. In an exemplary embodiment, there are 19 different serotypes.In one exemplary embodiment, there are 20 different serotypes. In one exemplary embodiment, there are 21 different serotypes. In one exemplary embodiment, there are 22 different serotypes. In one exemplary embodiment, there are 23 different serotypes. In one exemplary embodiment, there are 24 different serotypes. The capsular saccharide is conjugated to a carrier protein to form an immunogenic conjugate as described below. In one specific exemplary embodiment, the capsular saccharide of Streptococcus pneumoniae can be conjugated individually to the carrier protein. In another specific exemplary embodiment, the capsular saccharide can be conjugated directly to the carrier protein. 1.1 Carrier protein One component of the immunogenic conjugate described herein is a carrier to which the saccharide is covalently linked. Specifically, it IF-2019-79554467-APN-ANP#INPI Page 8 of 67 may use a carrier protein. In this description, the term carrier protein may be used interchangeably. Specifically, the carrier protein can be a protein that is non-toxic, does not induce reactions, and is available in sufficient quantity and purity. The carrier protein must be amenable to standard conjugation methods. In one exemplary embodiment, each capsular saccharide is conjugated to the same carrier protein (each capsular saccharide molecule is conjugated to a single carrier protein). In another exemplary embodiment, two or more capsular saccharides can be conjugated to a carrier protein (each capsular saccharide molecule is conjugated to a single carrier protein). In one exemplary embodiment, CRM197 is used as the carrier protein. The CRM197 protein is a non-toxic variant of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM197 is produced by Corynebacterium diphtheriae C7 (β197) grown in a yeast extract-casamino acid-based medium. Typically, CRM197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography. In another exemplary embodiment, tetanus toxoid (TT) can be used as the carrier protein. Other suitable carrier proteins include bacterial toxoids, for example, DT (diphtheria toxoid), TT (tetanus toxoid) fragment C, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, and exotoxin A derived from Pseudomonas aeruginosa. Bacterial outer membrane proteins, by IF-2019-79554467-APN-ANP#INPI Page 9 of 67 example, outer membrane complex c (OMPC), porin, transferrin-binding protein, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group B streptococcus-derived peptidase C5a, Haemophilus influenzae protein D, pneumococcal pneumolysin (including the layer detoxified in some way, e.g., dPLY-GMBS, dPLY-formaldehyde, or PhtX (including PhtA, PhtB, PhtD, PhtE, and Pht protein fusions, e.g., PhtDE fusions and PhtBE fusions)) may also be used. Other proteins, such as ovalbumin, California limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivatives (PPD) of tuberculin, PorB (from N.meningitidis), PD (Haemophilus influenzae protein D) or immunologically equivalent functional versions thereof, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, growth factors or hormones, artificial proteins containing multiple human CD4+ T cell epitopes of various pathogen-derived antigens, e.g. N19 protein, iron uptake protein, C. difficile toxin A or B and flagellin can also be used as a carrier protein. Other DT mutants, for example, diphtheria toxoid mutants CRM176, CRM228, CRM45, CRM9, CRM45, CRM102, CRM103 and CRM107, deletion or mutation of Glu 148 to Asp, Gln or Ser and / or deletion or mutation of Ala 158 to Gly and other known mutations may be used, mutation of at least one residue of Lys 516, Lys 526, Phe 530 and / or Lys 534 and other known mutations or fragments thereof. Specifically, CRM197 can be used as the carrier protein. Specifically, the carrier protein CRM197 can be covalently linked to the capsular saccharide described herein. IF-2019-79554467-APN-ANP#INPI Page 10 of 67 1.2 Capsular saccharide Throughout this description, the term saccharide may refer to a polysaccharide or an oligosaccharide and includes both. In most exemplary embodiments, the saccharide is a polysaccharide, particularly a capsular polysaccharide from Streptococcus pneumoniae. The capsular polysaccharide described herein can be prepared using a purification procedure known to those skilled in the art (e.g., see U.S. Patent Application Publication No. 2008 / 0286838, etc.). It can also be produced using synthetic protocols. In the present description, the capsular polysaccharide can be prepared, for example, from serotype 9N of Streptococcus pneumoniae. In another exemplary embodiment, an immunogenic conjugate and an immunogenic composition containing the capsular polysaccharide of the present description can be prepared using one or more serotypes from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, including serotype 9N. Typically, the capsular polysaccharide is prepared by proliferating each serotype of Streptococcus pneumoniae in a medium (e.g., a soy-based medium). The polysaccharide is obtained from bacterial culture. The Streptococcus pneumoniae bacterial strains used to prepare each polysaccharide used in the immunogenic protein-saccharide conjugate described herein may be obtained from established culture collections (e.g., strains deposited in ATCC or available from the Streptococcal Reference Laboratory of the Centers for Disease Control and Prevention (Atlanta, Georgia)) or clinical specimens. IF-2019-79554467-APN-ANP#INPI Page 11 of 67 In an exemplary embodiment, Streptococcus pneumoniae bacterial cells are cultured in a soy-based medium. The capsular polysaccharide is then purified using known purification techniques, including centrifugation, deep filtration, precipitation, ultrafiltration, activated carbon treatment, and diafiltration. The purified capsular polysaccharide is activated to enable reactivity and is then used to prepare the immunogenic conjugate. In one exemplary embodiment, the capsular saccharide of the present description may be an oligosaccharide. The oligosaccharide has a small number of repeating units (typically 5–15 repeating units) and is typically derived synthetically or by hydrolysis from a polysaccharide. Specifically, all capsular saccharides described herein and in the immunogenic composition herein may be polysaccharides. High molecular weight capsular polysaccharides are capable of inducing certain antibody immune responses due to the epitopes present on their antigenic surface. Specifically, the isolation and purification of the high molecular weight capsular polysaccharide are contemplated for use in the immunogenic conjugate and composition herein. In one exemplary embodiment, the capsular saccharide of serotype 9N purified before conjugation can be activated to have a molecular weight of 50-800 kDa, 80-780 kDa, 100-770 kDa, 120-760 kDa, 140-750 kDa, 150-740 kDa, 160-730 kDa, 170-735 kDa, 180-720 kDa, 190-710 kDa, 200-700 kDa, 220-690 kDa, 240-680 kDa, 260-670 kDa or 270-660 kDa. IF-2019-79554467-APN-ANP#INPI Page 12 of 67 When the 9N serotype saccharide has a molecular weight as described above, it can exhibit superior conjugation to a carrier protein, a low percentage of free saccharide, and superior immunogenicity. The carrier protein can form a conjugate with serotype 9N or with other serotypes besides serotype 9N. The serotypes can have the following molecular weights. Serotypes 1, 4, 6B, 7F and 11A can have a molecular weight of 100-2000 kDa, 200-1700 kDa, 300-1500 kDa, 400-1400 kDa or 600-1300 kDa. Serotypes 2, 3, 10A and 19F can have a molecular weight of 10-2000 kDa, 50-1700 kDa, 200-1500 kDa, 400-1200 kDa or 500-1000 kDa. Serotypes 5, 6A, 8, 18C, 20 and 23F can have a molecular weight of 100-2000 kDa, 200-1700 kDa, 300-1500 kDa, 400-1400 kDa or 200-1000 kDa. Serotypes 9V and 15B can have a molecular weight of 100-4000 kDa, 200-3500 kDa, 300-3000 kDa, 400-2800 kDa, 500-2600 kDa or 600-2500 kDa. Serotype 12F can have a molecular weight of 100-4000 kDa, 300-3500 kDa, 500-3000 kDa, 700-2800 kDa, 800-2600 kDa or 1000-2500 kDa. Serotype 14 can have a molecular weight of 500-4000 kDa, 800-3800 kDa, 1000-3500 kDa, 1200-3200 kDa or 1500-3000 kDa. Serotype 17F can have a molecular weight of 10-4000 kDa, 100-3500 kDa, 200-3000 kDa, 300-2000 kDa or 400-1500 kDa. Serotype 19A can have a molecular weight of 10-1500 kDa, 50-1000 kDa, 70-800 kDa, 80-700 kDa, 90-650 kDa or 100-600 kDa. Serotype 22F can have a molecular weight of 100-4000 kDa, 200-3900 kDa, 300-3800 kDa, 400-3700 kDa or 500-3500 kDa. IF-2019-79554467-APN-ANP#INPI Page 13 of 67 Serotype 33F can have a molecular weight of 100-4000 kDa, 200-3500 kDa, 300-3000 kDa, 400-2500 kDa or 500-2000 kDa. The capsular polysaccharide of Streptococcus pneumoniae may be slightly reduced in size during a normal purification procedure. Even in this case, the capsular polysaccharide meets the molecular weight range for the serotypes. Furthermore, as described herein, the capsular polysaccharide may undergo mechanical or chemical sizing. The molecular weight range described above may be understood as the molecular weight of the purified polysaccharide before conjugation. 1.2.1 Pneumococcal polysaccharide serotype 9N The saccharide of serotype 9N can be obtained directly from the bacteria using an isolation procedure known to those skilled in the art (e.g., see the methods described in U.S. Patent Application Publication No. 2006 / 0228380, etc.). In addition, the saccharide can be produced using synthetic protocols. The Streptococcus pneumoniae serotype 9N strain can be obtained from established culture collections (e.g., the Center for Disease Control and Prevention Streptococcus Reference Laboratory (Atlanta, Georgia)) or clinical specimens. The bacterial cell is cultured in a medium, specifically a soy-based medium. Following fermentation of the bacterial cell, which produces the capsular polysaccharide of Streptococcus pneumoniae serotype 9N, the bacterial cell is lysed to produce a cell lysate. The serotype 9N polysaccharide can then be isolated from the cell lysate using purification techniques known to the art, including centrifugation, deep filtration, IF-2019-79554467-APN-ANP#INPI Page 14 of 67 precipitation, ultrafiltration, activated carbon treatment, diafiltration, and / or column chromatography (see, for example, the methods described in U.S. Patent Application Publication No. 2006 / 0228380, etc.). The purified serotype 9N capsular polysaccharide can be used for the preparation of an immunogenic conjugate. The serotype 9N capsular polysaccharide obtained by purifying the serotype 9N polysaccharide from Streptococcus pneumoniae lysate and optionally sizing the purified polysaccharide can be characterized by different parameters, including, for example, the molecular weight (MW) of the serotype 9N capsular polysaccharide. In some exemplary embodiments, the purified polysaccharide obtained from Streptococcus pneumoniae serotype 9N before conjugation may have a molecular weight of 5–5,000 kDa. In one exemplary embodiment, the capsular polysaccharide may have a molecular weight of 50–1,000 kDa. In another exemplary embodiment, the capsular polysaccharide may have a molecular weight of 70–900 kDa. In yet another exemplary embodiment, the capsular polysaccharide may have a molecular weight of 100–800 kDa. In another embodiment, by way of example, the purified 9N serotype capsular saccharide can be activated prior to conjugation to have a molecular weight of 50-800 kDa, 80-780 kDa, 100-770 kDa, 120-760 kDa, 140-750 kDa, 150-740 kDa, 160-730 kDa, 170-735 kDa, 180-720 kDa, 190-710 kDa, 200-700 kDa, 220-690 kDa, 240-680 kDa, 260-670 kDa, 270-660 kDa or similar molecular weight ranges.Any whole number within any of the above ranges is considered as a way of realizing the present description. IF-2019-79554467-APN-ANP#INPI Page 15 of 67 The polysaccharide may slightly decrease in size during a normal purification procedure. Furthermore, as described herein, the polysaccharide may undergo sizing prior to conjugation. The molecular weight range mentioned above refers to that of the purified polysaccharide after the final sizing step (e.g., after purification, hydrolysis, and activation) prior to conjugation. 1.3 Carrier protein-capsular saccharide conjugate The purified polysaccharide is chemically activated to enable it to react with a carrier protein. The activated saccharide is then conjugated with the carrier protein to form a saccharide-protein conjugate. The chemical activation of the polysaccharide and its subsequent conjugation with the carrier protein can be achieved using the activation and conjugation methods described herein. (1) Conjugation method In one exemplary embodiment, for conjugation, the saccharide can be activated using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide can then be coupled directly or via a spacer (linker) group to an amino group in the carrier protein. For example, the spacer can be cystamine or cysteamine to provide a thiolated polysaccharide, which can be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimide [e.g., ethyl iodoacetimide HCl], N-bromoacetate). IF-2019-79554467-APN-ANP#INPI Page 16 of 67 succinimidyl, SIAB, SIA or SBAP). Specifically, the cyanate ester (optionally prepared by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derived saccharide is conjugated to the carrier protein using carbodiimide chemistry (e.g., EDAC or EDC) via a carboxyl group on the carrier protein. Other suitable conjugation techniques utilize carbodiimides, hydrazides, active ester, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Conjugation may involve a carbonyl linker, which can be formed by the reaction of a free hydroxyl group on the saccharide with CDI followed by reaction with a protein to form a carbamate linkage. This may involve the reduction of the anomeric terminus to a primary hydroxyl group, the optional protection / deprotection of the primary hydroxyl group, the reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and the coupling of the CDI carbamate intermediate with an amino group on a protein. (2) Reductive amination In one specific exemplary embodiment, the capsular polysaccharide of Streptococcus pneumoniae is conjugated to the carrier protein by reductive amination. Reductive amination involves the activation of the polysaccharide by reaction with an oxidizing agent and the conjugation of the activated polysaccharide to the carrier protein by reduction. Reductive amination involves two steps: (1) activation of the polysaccharide by reaction with the oxidizing agent and (2) conjugation of the activated polysaccharide to the carrier protein by reductive amination. Specifically, before the IF-2019-79554467-APN-ANP#INPI Page 17 of 67 reaction with the oxidizing agent, the polysaccharide can optionally be sized by chemical hydrolysis using acetic acid or hydrochloric acid. (2-1) Activation of the polysaccharide by reaction with an oxidizing agent In one specific exemplary embodiment, the oxidizing agent is periodate. Periodate randomly oxidizes the adjacent hydroxyl group of a carbohydrate to form a reactive aldehyde group and causes the cleavage of a C-C bond. The term periodate includes both periodate and periodic acid. This term also includes both metaperiodate (IO₄⁻) and orthoperiodate (IO₅⁻). The term periodate also includes several periodate salts, including sodium periodate and potassium periodate. In one specific exemplary embodiment, the saccharide can be oxidized in the presence of sodium metaperiodate. Periodate can be used in an amount of approximately 0.03-0.17 μg, specifically 0.025-0.18 μg, more specifically 0.02-0.19 μg, per 1 μg of saccharide. Saccharide can be activated as desired within the above range. Outside of this range, the effect may be unsatisfactory. In one exemplary embodiment, the activated capsular saccharide is purified. The activated capsular saccharide is purified according to methods known to those skilled in the art, for example, gel permeation chromatography (GPC), dialysis, or ultrafiltration / diafiltration. Specifically, the activated saccharide is purified by ultrafiltration / diafiltration using an ultrafiltration device. Activated saccharide can be characterized by a degree of oxidation and molecular weight. IF-2019-79554467-APN-ANP#INPI Page 18 of 67 In a specific exemplary embodiment, the activated 9N serotype polysaccharide can have an oxidation degree of 0.5-25, 0.6-23, 0.821, 1-20.8, 1.1-20.5, 1.2-20.3, 1.3-20, 1.4-19.5, 1.5-19.3, 1.6-19.2, 1.7-19.1 or 2-19. When the degree of oxidation is within the range mentioned above, the conjugate can be formed stably with high yield. Furthermore, the proportion of free saccharide can be reduced. After the oxidation step of the saccharide, the activated saccharide and the carrier protein can be lyophilized (freeze-dried) either independently (separate lyophilization) or together (co-lyophilization). In one exemplary embodiment, the activated saccharide and the carrier protein are co-lyophilized. In another exemplary embodiment, the activated saccharide and the carrier protein are lyophilized independently. In one exemplary embodiment, freeze-drying is carried out in the presence of a cryoprotectant / drying protectant. In one exemplary embodiment, the cryoprotectant / drying protectant is a saccharide. In one specific exemplary embodiment, the saccharide is selected from sucrose, trehalose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinite. In one specific exemplary embodiment, the saccharide is sucrose. (2-2) Formation of the polysaccharide-carrier protein conjugate by reaction with a reducing agent Reduction The second step in the conjugation process via reductive amination is the reduction of the activated polysaccharide and the carrier protein using a reducing agent to form the conjugate. Reducing agents IF-2019-79554467-APN-ANP#INPI Page 19 of 67. Suitable reducing agents include cyanoborohydride, for example, sodium cyanoborohydride, boranepyridine, or borohydride exchange resin. In one specific embodiment, the reducing agent is sodium cyanoborohydride. In one embodiment, the reduction reaction is carried out in an aqueous solvent. In another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide). The DMSO or DMF solvent can be used to reconstitute the lyophilized activated polysaccharide and carrier protein. In one exemplary embodiment, the reducing agent may be sodium cyanoborohydride, sodium triacetoxyborohydride, or sodium or zinc borohydride in the presence of a Brønsted or Lewis acid, or amine boranes such as pyridine borane, 2-picolin borane, 2,6-diboranemethanol, dimethylamine borane, t-BuMeiPrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB). In a specific exemplary embodiment, the reducing agent is sodium cyanoborohydride. Capping At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate. These can be protected using a suitable protecting agent (inactivation). In one exemplary embodiment, the protecting agent is sodium borohydride (NaBH4). Purification After conjugation (reduction and, optionally, protection) of the saccharide and carrier protein, the saccharide-protein capsular conjugate can be purified by various techniques known to experts in the IF-2019-79554467-APN-ANP#INPI Page 20 of 67. These techniques include ultrafiltration / diafiltration, tangential flow filtration, precipitation / elution, ion-exchange chromatography / size-exclusion chromatography, and deep filtration. In one exemplary embodiment, the saccharide-protein conjugate is purified by a combination of ultrafiltration / diafiltration and either ion-exchange chromatography or size-exclusion chromatography. In another exemplary embodiment, the saccharide-protein conjugate is subjected to sterile filtration. The saccharide-protein conjugate can be characterized by its molecular weight determined by SEC-MALLS, the saccharide-to-carrier-protein ratio (weight / weight), the free saccharide content, the free protein content, and the molecular size distribution (Kd). (3) Characterization of the conjugate. In one exemplary embodiment, the conjugate may have a molecular weight of 100–10,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 200–9,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 300–8,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 400–7,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 500–6,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 600–5,000 kDa. In one exemplary embodiment, the conjugate may have a molecular weight of 500–4,000 kDa. Any whole number within any of the above ranges is considered an embodiment of the present description. IF-2019-79554467-APN-ANP#INPI Page 21 of 67 When the molecular weight is within the range mentioned above, the conjugate can be formed stably with high yield. Furthermore, the proportion of free saccharide can be reduced. In addition, superior immunogenicity can be achieved within the same molecular weight range. Once the individual saccharide-protein conjugates are purified, they are combined to formulate the immunogenic composition described herein. The saccharide-protein conjugates of the serotypes in this description can be characterized by a ratio of the saccharide to the carrier protein (amount of saccharide / amount of protein, weight / weight). In some exemplary embodiments, the (w / w) ratio of the saccharide to the carrier protein in the saccharide-protein conjugate for each serotype is 0.5-2.5, 0.4-2.3, 0.3-2.1, 0.24-2, 0.2-1.8, 0.18-1.6, 0.16-1.4, 0.14-1.2, 0.12-1 or 0.1-1 (e.g., approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3 about 2.4 or about 2.5). Specifically, the carrier protein may be CRMi97. When the ratio of saccharide to carrier protein is within the specified range, the conjugate can be formed stably with high yield. Furthermore, the proportion of free saccharide can be reduced. IF-2019-79554467-APN-ANP#INPI Page 22 of 67 Furthermore, superior immunogenicity can be achieved and the conjugate can be kept stable without interference from other serotypes within the above range. The conjugate and immunogenic composition described herein may contain a free saccharide that is not covalently conjugated to the carrier protein but is nevertheless present in the saccharide-protein conjugate composition. The free saccharide may be non-covalently associated with the saccharide-protein conjugate (i.e., non-covalently bound, adsorbed, or trapped in or by the saccharide-protein conjugate). In a specific exemplary embodiment, the saccharide-protein conjugate contains less than approximately 60%, approximately 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 60% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 50% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype.In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 40% of a free polysaccharide from each serotype based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 30% of a free polysaccharide from each serotype based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the conjugate. IF-2019-79554467-APN-ANP#INPI Page 23 of 67. The saccharide-protein conjugate of each serotype contains less than approximately 25% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 20% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 15% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. In a specific exemplary embodiment, the saccharide-protein conjugate of each serotype contains less than approximately 10% of a free polysaccharide from each serotype, based on the total amount of polysaccharide from each serotype. The saccharide-protein conjugate of each serotype can also be characterized by its molecular size distribution (Kd). A size exclusion chromatography medium (CL-4B; crosslinked agarose beads, 4%) can be used to determine the relative molecular size distribution of the conjugate. Size exclusion chromatography (SEC) is used on a gravity-fed column to profile the molecular size distribution of the conjugate. Large molecules excluded from the pores in the medium elute more rapidly than small molecules. A fraction collector is used to collect the eluate from the column. The fractions are then assayed colorimetrically using a saccharide assay. For Kd determination, the column is calibrated to establish the fraction at which molecules are completely excluded (V0; Kd = 0) and the fraction representing maximum retention (Vi; Kd = 1). The fraction at which a IF-2019-79554467-APN-ANP#INPI Page 24 of 67 specific sample attribute (Ve) is related to Kd by the expression Kd= (Ve - Vo) / (Ví - Vo). In one specific exemplary embodiment, at least 15% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or lower on a CL-4B column. In a specific exemplary embodiment, at least 20% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or less on a CL-4B column. In a specific exemplary embodiment, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or less on a CL-4B column. In a specific exemplary embodiment, at least 60% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or less on a CL-4B column. In a specific exemplary embodiment, at least 50-80% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or lower on a CL-4B column. In a specific exemplary embodiment, at least 65-80% of the saccharide-protein conjugate of each serotype may have a Kd of 0.3 or lower on a CL-4B column.In one specific exemplary embodiment, at least 15-60% of the saccharide-protein conjugate of each serotype can have a Kd of 0.3 or lower on a CL-4B column. 1.4 Combination of capsular saccharide-carrier protein conjugate In one exemplary embodiment, the immunogenic composition described herein contains a saccharide-protein conjugate of Streptococcus pneumoniae serotype 9N. In another exemplary embodiment, it may contain one, two, or three saccharide-protein conjugates selected from a group that IF-2019-79554467-APN-ANP#INPI Page 25 of 67 consists of a saccharide-protein conjugate of Streptococcus pneumoniae serotype 2, a saccharide-protein conjugate of Streptococcus pneumoniae serotype 17F, and a saccharide-protein conjugate of Streptococcus pneumoniae serotype 20. In an exemplary embodiment, any of the immunogenic composition may further contain a saccharide-protein conjugate of one or more selected from a group consisting of serotypes 8, 10A, 11A, and 15B of Streptococcus pneumoniae. In an exemplary embodiment, any of the immunogenic composition may further contain a saccharide-protein conjugate of one or more selected from a group consisting of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F of Streptococcus pneumoniae. In an exemplary embodiment, any of the immunogenic composition may further contain a saccharide-protein conjugate of one or more selected from a group consisting of serotypes 1, 5, 6A, 7F, and 19A of Streptococcus pneumoniae. In an exemplary embodiment, any of the immunogenic compositions may further contain a saccharide-protein conjugate of Streptococcus pneumoniae serotype 3. In one specific exemplary embodiment, all saccharide-protein conjugates of the immunogenic composition are individually conjugated to the carrier protein. In an exemplary embodiment, any of the saccharide-protein conjugates of the immunogenic composition are individually conjugated with CRM197 or TT. IF-2019-79554467-APN-ANP#INPI Page 26 of 67 In one exemplary embodiment, the immunogenic composition contains 8–24 different serotypes of Streptococcus pneumoniae. In one exemplary embodiment, the immunogenic composition may contain saccharide-protein conjugates of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 different serotypes. In one exemplary embodiment, the immunogenic composition may contain saccharide-protein conjugates of 16 or 20 different serotypes. 2. Administration of immunogenic composition dosage The amount of capsular saccharide-carrier protein conjugate(s) in each dose is selected as an amount that induces an immunoprotective response without significant side effects in typical vaccines. This amount will vary depending on the specific immunogen used and how it is administered. 2.1 Amount of capsular saccharide-carrier protein conjugate The amount of a particular capsular saccharide-carrier protein conjugate in an immunogenic composition can be calculated based on the total polysaccharide (conjugated and unconjugated) in the conjugate. For example, a capsular saccharide-carrier protein conjugate with 20% free polysaccharide will have approximately 80 pg of conjugated polysaccharide and approximately 20 pg of unconjugated polysaccharide in 100 pg of polysaccharide. The amount of saccharide-protein conjugate can vary depending on the pneumococcal serotype. The saccharide concentration can be measured using a uronic acid or anthrone assay. IF-2019-79554467-APN-ANP#INPI Page 27 of 67 The immunogenic amount of different polysaccharide components in the immunogenic composition can be diverse, and each may contain approximately 1 page, approximately 2 pages, approximately 3 pages, approximately 4 pages, approximately 5 pages, approximately 6 pages, approximately 7 pages, approximately 8 pages, approximately 9 pages, approximately 10 pages, approximately 15 pages, approximately 20 pages, approximately 30 pages, approximately 40 pages, approximately 50 pages, approximately 60 pages, approximately 70 pages, approximately 80 pages approximately 90 μg or approximately 100 μg of any particular polysaccharide antigen. In general, each dose may contain 0.1–100 μg of polysaccharide for a given serotype, more particularly 0.5–20 μg, more particularly 1.0–10 μg, and even more particularly 2.0–5.0 μg. Any whole number within any of the above ranges is considered a form of realization of the present description. In an exemplary embodiment, each dose may contain approximately 1.0 μg, approximately 1.6 μg, approximately 2.2 μg, approximately 2.8 μg, approximately 3.4 μg, approximately 4.0 μg, approximately 4.6 μg, approximately 5.2 μg, approximately 1.2 μg, approximately 1.8 μg, approximately 2.4 μg, approximately 3.0 μg, approximately 3.6 μg, approximately 4.2 μg, approximately 4.8 μg, approximately 5.4 μg, approximately 1.4 μg, approximately 2.0 μg, approximately 2.6 μg, approximately 3.2 μg, approximately 3.8 μg, approximately 4.4 μg, approximately 5.0 μg, approximately 5.6 μg, IF-2019-79554467-APN-ANP#INPI Page 28 of 67 approximately 5.8 μ0 or approximately 6.0 μ0 of polysaccharide for each particular capsular saccharide-carrier protein conjugate. In an exemplary embodiment, each dose may contain approximately 1.1 μg, approximately 1.2 μg, approximately 1.3 μg, approximately 1.4 μg, approximately 1.5 μg, approximately 1.6 μg, approximately 1.7 μg, approximately 1.8 μg, approximately 1.9 μg, approximately 2.0 μg, approximately 2.1 μg, approximately 2.2 μg, approximately 2.3 μg, approximately 2.4 μg, approximately 2.5 μg, approximately 2.6 μg, approximately 2.7 μg, approximately 2.8 μg, approximately 2.9 μg, or approximately 3.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F. In an exemplary embodiment, each dose may contain approximately 2.0, approximately 2.6, approximately 3.2, approximately 3.8, approximately 4.4, approximately 5.0 μg, approximately 2.2 μg, approximately 2.8 μg, approximately 3.4 μg, approximately 4.0 μg, approximately 4.6 μg, approximately 5.2 μg, approximately 2.4 μg, approximately 3.0 μg, approximately 3.6 μg, approximately 4.2 μg, approximately 4.8 μg, approximately 5.4 μg, approximately 5.6 μg, approximately 5.8 μg, or approximately 6.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In one exemplary embodiment, each dose can contain approximately 1.5-3.0 μg of polysaccharide for the saccharide conjugate IF-2019-79554467-APN-ANP#INPI Page 29 of 67 Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F protein and approximately 3.0-6.0 μ0 of polysaccharide for the saccharidoprotein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 2.0-2.5 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F and about 4.0-4.8 μg of polysaccharide for the saccharide-protein conjugate of Stubotococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 2.2 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F and approximately 4.4 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 1.1 μg, approximately 1.2 μg, approximately 1.3 μg, approximately 1.4 μg, approximately 1.5 μg, approximately 1.6 μg, approximately 1.7 μg, approximately 1.8 μg, approximately 1.9 μg, approximately 2.0 μg, approximately 2.1 μg, approximately 2.2 μg, approximately 2.3 μg, approximately 2.4 μg, approximately 2.5 μg, approximately 2.6 μg, approximately 2.7 μg, approximately 2.8 μg, approximately 2.9 μg, or approximately 3.0 μg of polysaccharide for the IF-2019-79554467-APN-ANP#INPI Page 30 of 67 saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F. In an exemplary embodiment, each dose may contain approximately 2.0, approximately 2.6, approximately 3.2, approximately 3.8, approximately 4.4, approximately 5.0 μg, approximately 2.2 μg, approximately 2.8 μg, approximately 3.4 μg, approximately 4.0 μg, approximately 4.6 μg, approximately 5.2 μg, approximately 2.4 μg, approximately 3.0 μg, approximately 3.6 μg, approximately 4.2 μg, approximately 4.8 μg, approximately 5.4 μg, approximately 5.6 μg, approximately 5.8 μg, or approximately 6.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 1.5-3.0 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and approximately 3.0-6.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 2.0-2.5 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and approximately 4.0-4.8 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. IF-2019-79554467-APN-ANP#INPI Page 31 of 67 In an exemplary embodiment, each dose may contain approximately 2.2 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9N, 9V, 10A, 11A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and approximately 4.4 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 1.1 μg, approximately 1.2 μg, approximately 1.3 μg, approximately 1.4 μg, approximately 1.5 μg, approximately 1.6 μg, approximately 1.7 μg, approximately 1.8 μg, approximately 1.9 μg, approximately 2.0 μg, approximately 2.1 μg, approximately 2.2 μg, approximately 2.3 μg, approximately 2.4 μg, approximately 2.5 μg, approximately 2.6 μg, approximately 2.7 μg, approximately 2.8 μg, approximately 2.9 μg, or approximately 3.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F. In an exemplary embodiment, each dose may contain approximately 2.0, approximately 2.6, approximately 3.2, approximately 3.8, approximately 4.4, approximately 5.0 μg, approximately 2.2 μg, approximately 2.8 μg, approximately 3.4 μg, approximately 4.0 μg, approximately 4.6 μg, approximately 5.2 μg, approximately 2.4 μg, approximately 3.0 μg, approximately 3.6 μg, approximately 4.2 μg, approximately 4.8 μg, approximately 5.4 μg, approximately 5.6 μg, approximately 5.8 μg, or approximately 6.0 μg of IF-2019-79554467-APN-ANP#INPI Page 32 of 67 polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 1.5-3.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and approximately 3.0-6.0 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 2.0-2.5 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and about 4.0-4.8 μg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. In an exemplary embodiment, each dose may contain approximately 2.2 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and / or 33F and approximately 4.4 pg of polysaccharide for the saccharide-protein conjugate of Streptococcus pneumoniae serotype 6B. 2.2 Amount of carrier protein Each dose may contain 10-150 μg of the carrier protein, in particular 15-100 μg of the carrier protein, more particularly 25-75 μg of the carrier protein, even more particularly 40-60 μg of the protein IF-2019-79554467-APN-ANP#INPI Page 33 of 67 carrier, for the carrier-saccharide conjugate composition of serotype 9N. In one exemplary embodiment, the carrier protein is CRM197. In a form of approximately 25 μg, approximately 28 μg, approximately 31 μg, approximately 34 μg, approximately 37 μg, approximately 40 μg, approximately 43 μg, approximately 46 μg, approximately 49 μg, approximately 52 μg, approximately 55 μg, approximately 58 μg, approximately 61 μg, approximately 64 μg, approximately 67 μg, approximately 70 μg, exemplary realization, approximately 26 approximately 29 approximately 32 approximately 35 approximately 38 approximately 41 approximately 44 approximately 47 approximately 50 approximately 53 approximately 56 approximately 59 approximately 62 approximately 65 approximately 68 approximately 71 each dose may contain μg, approximately 27 μg, μg, approximately 30 μg, μg, approximately 33 μg, μg, approximately 36 μg, μg, approximately 39 μg, μg, approximately 42 μg, μg, approximately 45 μg, μg, approximately 48 μg, μg, approximately 51 μg, μg,approximately 54 μg, μg, approximately 57 μg, μg, approximately 60 μg, μg, approximately 63 μg, μg, approximately 66 μg, μg, approximately 69 μg, μg, approximately 72 μg, approximately 73 μg, approximately 74 μg or approximately 75 μg of the carrier protein. In one exemplary embodiment, the carrier protein is CRM197. 3. Additional antigens The immunogenic composition of the present description contains a conjugated saccharin antigen of Streptococcus pneumoniae (saccharin conjugate IF-2019-79554467-APN-ANP#INPI Page 34 of 67 100 protein). The composition may also contain antigens from other pathogens, particularly bacteria and / or viruses. Specifically, the additional antigens are selected from diphtheria toxoid (D), tetanus toxoid (T), pertussis antigen (P), which is typically acellular surface antigen (Pa), hepatitis B virus (HBV) (HBsAg), hepatitis A virus (HAV) antigen, Haemophilus influenzae type b conjugate capsular saccharide (Hib), and inactivated polio vaccine (IPV). In one exemplary embodiment, the immunogenic composition described herein contains DT-Pa. In one exemplary embodiment, the immunogenic composition described herein contains DT-Pa-Hib, DT-Pa-IPV, or DT-Pa-HBsAg. In one exemplary embodiment, the immunogenic composition described herein contains DT-Pa-HBsAg-IPV or DT-Pa-HBsAg-Hib. In one exemplary embodiment, the immunogenic composition described herein contains DT-Pa-HBsAg-IPV-Hib. Pertussis antigens Bordetella pertussis causes pertussis (whooping cough). Pertussis antigens in vaccines are either cellular (whole cells, in the form of inactivated Bordetella pertussis cells) or acellular. The preparation of cellular pertussis antigens is well documented (for example, antigens can be obtained by heat inactivation of phase I Bordetella pertussis cultures). Specifically, however, acellular antigens are used in this description. When acellular antigens are used, one, two, or (specifically) three of the following antigens may be used: (1) detoxified pertussis toxin (pertussis toxoid or PT); (2) filamentous hemagglutinin (FHA); (3) pertactin (also known as 69 kDa outer membrane protein). IF-2019-79554467-APN-ANP#INPI Page 35 of 67 101 FHA and pertactin may be treated with formaldehyde prior to use according to this description. Specifically, PT is detoxified by treatment with formaldehyde and / or glutaraldehyde. Specifically, acellular pertussis antigens are adsorbed onto one or more aluminum salt adjuvants. Alternatively, they may be added in a non-adsorbed state. When pertactin is added, it may be pre-adsorbed onto an aluminum hydroxide adjuvant. Specifically, all PT, FHA, and pertactin may be adsorbed onto an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant. More specifically, all PT, FHA, and pertactin may be adsorbed onto aluminum hydroxide. Inactivated poliovirus vaccines: Poliovirus causes poliomyelitis. In one specific embodiment of the present description, IPV can be used instead of the oral poliovirus vaccine. Before administration to patients, the polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde. Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically different, and infection with one type does not protect against infection with the others. Therefore, three poliovirus antigens are preferred in this description: poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-1 strain), and poliovirus type 3 (e.g., Saukett strain). Specifically, the viruses are individually cultured, purified, and inactivated, and then combined to give a bulk trivalent mixture for use in this description. IF-2019-79554467-APN-ANP#INPI Page 36 of 67 102 Diphtheria toxoid Corynebacterium diphtheriae causes diphtheria. The diphtheria toxin can be treated (for example, using formalin or formaldehyde) to eliminate its toxicity while preserving its ability to induce specific antibodies against the toxin after injection. These diphtheria toxoids are used in diphtheria vaccines. Specifically, the diphtheria toxoid is prepared by formaldehyde treatment. It can be obtained by culturing Corynebacterium diphtheriae in a growth medium, followed by formaldehyde treatment, ultrafiltration, and precipitation. The toxoid material can then be treated by a process that includes sterile filtration and / or dialysis. Specifically, the diphtheria toxoid is adsorbed onto an aluminum hydroxide adjuvant. Tetanus toxoid Clostridium tetani causes tetanus. The tetanus toxin can be processed to produce a protective toxoid. This toxoid is used in tetanus vaccines. Specifically, tetanus toxoids are prepared by formaldehyde treatment. Tetanus toxoid can be obtained by culturing Clostridium tetani in a growth medium, followed by formaldehyde treatment, ultrafiltration, and precipitation. The material can then be treated using a process that includes sterile filtration and / or dialysis. Hepatitis A virus antigens: The hepatitis A virus (HAV) is one of the known agents that cause viral hepatitis. A preferred HAV formulation is based on inactivated virus, and inactivation can be achieved through formalin treatment. IF-2019-79554467-APN-ANP#INPI Page 37 of 67 103 The hepatitis B virus (HBV) is one of the known agents that causes viral hepatitis. The main component of the capsid is a protein known as HBV surface antigen, or more commonly, HBsAg, which is typically a 226-amino-acid polypeptide with a molecular weight of approximately 24 kDa. All existing hepatitis B vaccines contain HBsAg, and when this antigen is administered in a standard vaccine, it stimulates the production of anti-HBsAg antibodies that protect against HBV infection. For vaccine preparation, HBsAg has been prepared in two ways: purification of the antigen in particulate form from the plasma of chronic hepatitis B carriers or expression of the protein using recombinant DNA methods (e.g., recombinant expression in yeast cells). Unlike native HBsAg (i.e., as in the plasma-purified product), yeast-expressed HBsAg is generally not glycosylated, and this is the preferred form of HBsAg for use in this description. Haemophilus influenzae type b conjugated antigens: Haemophilus influenzae type b (Hib) causes bacterial meningitis. Hib vaccines are typically based on the capsular saccharide antigen, the preparation of which is well documented. The Hib saccharide can be conjugated to a carrier protein to enhance its immunogenicity, especially in children. Typical carrier proteins include tetanus toxoid, diphtheria toxoid, CRM197, Haemophilus influenzae protein D, and an outer membrane protein complex from serogroup B meningococcus. The saccharide moiety of the conjugate may contain full-length polyribosylribitol phosphate (PRP) prepared from IF-2019-79554467-APN-ANP#INPI Page 38 of 67 104 Hib bacteria and / or full-length PRP fragments. Hib conjugates may or may not be adsorbed to an aluminum salt adjuvant. In an exemplary embodiment, the immunogenic composition of the present description may further contain a conjugated capsular saccharide of Neisseria meningitidis serogroup Y (MenY) and / or a conjugated capsular saccharide of Neisseria meningitidis serogroup C (MenC). In an exemplary embodiment, the immunogenic composition of the present description may further contain a conjugated capsular saccharide of Neisseria meningitidis serogroup A (MenY), a conjugated capsular saccharide of Neisseria meningitidis serogroup W135 (MenW135), a conjugated capsular saccharide of Neisseria meningitidis serogroup Y (MenY) and / or a conjugated capsular saccharide of Neisseria meningitidis serogroup C (MenC). In an exemplary embodiment, the immunogenic composition of the present description may further contain a conjugated capsular saccharide of Neisseria meningitidis serogroup W135 (MenW135), a conjugated capsular saccharide of Neisseria meningitidis serogroup Y (MenY) and / or a conjugated capsular saccharide of Neisseria meningitidis serogroup C (MenC). 4. Adjuvant In some exemplary embodiments, the immunogenic composition described herein may further contain at least one adjuvant. The term adjuvant refers to a compound or mixture that enhances the immune response to an antigen. The adjuvant may enhance the immune response to an antigen that exhibits weak immunogenicity when administered alone and / or increase the antibody titer to an antigen and / or reduce the dose of an effective antigen required to elicit an immune response in a subject. IF-2019-79554467-APN-ANP#INPI Page 39 of 67 105 For example, by inducing a weak antibody titer or a cell-mediated immune response. Therefore, the adjuvant generally serves to enhance the immune response, as experts in the field are well aware. Examples of suitable adjuvants that improve the effectiveness of the formulation include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) Oil-in-water emulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing varying amounts of MTP-PE) and formulated into submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics, Newton, Mass., USA), (b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, (c) Ribi Adjuvant System ™ (RAS; Corixa, Hamilton, Mont., USA) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components from a group consisting of monophospholipid A 3-O-desylated (MPL™), trehalose dimycolate (TDM) and cell wall skeleton (CWS), specifically MPL + CWS (Detox™); (3) saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, Mass., USA) or particles generated from them, for example, ISCOM (immunostimulatory complex formed by a IF-2019-79554467-APN-ANP#INPI Page 40 of 67 106 combination of cholesterol, saponins, phospholipids and amphiphilic proteins) and Iscomatrix (essentially the same as ISCOM, but without the proteins); (4) bacterial lipopolysaccharides and synthetic lipid A analogues (e.g. aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogues thereof, which are available from Corixa; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3O[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion); (5) synthetic polynucleotides such as oligonucleotides containing one or more CpG motifs; (6) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., interferon gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; and (7) complements such as the trimer of complement component C3d. In another exemplary embodiment, the adjuvant can be a mixture consisting of two, three or more of the above adjuvants, e.g., SBAS2 (oil-in-water emulsion containing monophosphoryl lipid A 3-desylated and QS21). IF-2019-79554467-APN-ANP#INPI Page 41 of 67 107 Muramyl peptides include N-acetylmuramil-L-threonineyl-disoglutamine (thr-MDP), N-acetylmuramil-L-alanine-2-(1'-2'-dipalmitoyl-snglycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc., although they are not limited to them. In one specific exemplary embodiment, the adjuvant is an aluminum salt. The aluminum salt adjuvant can be either a precipitated alum vaccine or an adsorbed alum vaccine. Aluminum salt adjuvants are well known in the art. Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel, Superfos, Amphogel, aluminum(III) hydroxide, aluminum hydroxyphosphate sulfate (aluminum phosphate adjuvant (APA)), amorphous alumina, alumina trihydrate, or aluminum trihydroxide. APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate the aluminum hydroxyphosphate. After mixing, the desired aggregate particles, ranging in size from 2 to 8 µm, are obtained by reducing the particle size using a high-shear mixer. The product is then diafiltered in physiological saline solution and subsequently steam sterilized. In one specific exemplary embodiment, commercially available Al(OH)3 (e.g., Alhydrogel or Superfos from Denmark / Accurate Chemical and Scientific Co. (Westbury, NY, USA)) is used to adsorb proteins at a ratio of 50–200 g of protein / mg of aluminum hydroxide. In another exemplary embodiment, protein adsorption depends on the pI (isoelectric pH) of the protein and the pH of the medium. A protein with a lower pI adsorbs to the positively charged aluminum ion more readily. IF-2019-79554467-APN-ANP#INPI Page 42 of 67 108 strength than a protein with a higher pI. Aluminum salts can establish an Ag depot that is slowly released over a period of 2-3 weeks, are involved in non-specific macrophage activation and complement activation and / or stimulate the innate immune mechanism (possibly through uric acid stimulation). In one specific exemplary embodiment, the adjuvant is a nucleotide sequence containing CpG, for example, a CpG-containing oligonucleotide, in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In another exemplary embodiment, the adjuvant is ODN 1826, which can be purchased from Coley Pharmaceutical Group. CpG-containing nucleotide, CpG-containing oligonucleotide, CpG oligonucleotide, and similar terms refer to a nucleotide molecule of 6–50 nucleotides in length containing an unmethylated CpG residue. In another exemplary embodiment, any other definition of the terms accepted in the art is also intended. CpG-containing oligonucleotide includes oligonucleotides modified using any synthetic internucleosidic linkage, modified bases, and / or modified sugars. The methods for using the CpG oligonucleotide are well known in the technique. In one specific exemplary embodiment, the adjuvant is an aluminum-based adjuvant selected from a group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one exemplary embodiment, the immunogenic composition described herein contains the aluminum phosphate adjuvant. 5. Formulation IF-2019-79554467-APN-ANP#INPI Page 43 of 67 109 The immunogenic composition described herein can be formulated in liquid form (i.e., solutions or suspensions) or in lyophilized form. Liquid formulations can be advantageously administered directly from their packaged form and are therefore ideal for injection without the need for reconstitution in an aqueous medium, as is otherwise required for the lyophilized compositions described herein. The formulation of the immunogenic composition described herein can be carried out using recognized methods in the art. For example, the individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle for preparing the composition. Examples of such vehicles include, but are not limited to, water, buffered saline solution, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solution. The present description provides an immunogenic composition containing any of the combinations of saccharide-protein conjugates described herein and a pharmaceutically acceptable excipient, carrier, or diluent. In an exemplary embodiment, the immunogenic composition of the present description is in the form of a liquid, specifically in the form of an aqueous liquid. The immunogenic composition of the present description may contain one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, an antioxidant such as a free radical scavenger or a chelating agent, or any multiple combination thereof. IF-2019-79554467-APN-ANP#INPI Page 44 of 67 110 In one exemplary embodiment, the immunogenic composition described herein contains a buffer. In one exemplary embodiment, the buffer has a pKa of approximately 3.5–7.5. In some exemplary embodiments, the buffer is phosphate, succinate, histidine, or citrate. In certain exemplary embodiments, the buffer is succinate at a final concentration of 1–10 mM. In one particular exemplary embodiment, the final concentration of the succinate buffer is approximately 5 mM. In one exemplary embodiment, the immunogenic composition described herein contains a salt. In some exemplary embodiments, the salt is selected from a group consisting of magnesium chloride, potassium chloride, sodium chloride, and a combination thereof. In one particular exemplary embodiment, the salt is sodium chloride. In one particular exemplary embodiment, the immunogenic composition described herein contains 150 mM sodium chloride. In an exemplary embodiment, the immunogenic composition described herein contains a surfactant. The surfactant is selected from one or more nonionic surfactants consisting of polyoxyethylene sorbitan fatty acid ester, polysorbate 80 (Tween 80), polysorbate 60 (Tween 60), polysorbate 40 (Tween 40), polysorbate 20 (Tween 20), polyoxyethylene alkyl ether (including, but not limited to, Brij 58 and Brij 35), as well as other materials such as Triton X-100, Triton X-114, NP40, Span 85, and nonionic surfactants of the Pluronic series (e.g., Pluronic 121), although not limited to the same. In a specific exemplary embodiment, the immunogenic composition contains polysorbate 80 or polysorbate 40, specifically polysorbate 80. In a specific exemplary embodiment, the immunogenic composition contains IF-2019-79554467-APN-ANP#INPI Page 45 of 67 111 polysorbate 80 at a concentration of approximately 0.001% to approximately 2% (with up to approximately 0.005% being preferred). In one exemplary embodiment, a container of the present description is made of glass, metals (e.g., steel, stainless steel, aluminum, etc.) and / or polymers (e.g., thermoplastics, elastomers, or thermoplastic elastomers). In one exemplary embodiment, the container of the present description is made of glass. In one exemplary embodiment, the present description provides a syringe filled with any of the immunogenic compositions described herein. In one particular exemplary embodiment, the syringe is silicone-coated and / or made of glass. 6. Use In an exemplary embodiment, the immunogenic composition described herein is for use as a medicament. The amount of conjugate in the formulation is selected as the quantity that induces an immunoprotective response without significant adverse effects. This amount will vary depending on the specific pneumococcal serotype. The immunogenic composition described herein can be used in various therapeutic or prophylactic methods to prevent, treat, or improve a bacterial infection, disease, or condition in a subject. In particular, the immunogenic composition described herein can be used to prevent, treat, or improve an infection, disease, or condition caused by Streptococcus pneumoniae in a subject. IF-2019-79554467-APN-ANP#INPI Page 46 of 67 112 All references or patent applications cited in this description are incorporated herein by reference. The following examples illustrate this description. These examples are performed using standard techniques that are well-known and routine for those skilled in the art, except where otherwise described in detail. The examples are illustrative but do not limit this description. In one aspect, the present description provides a method for preventing, treating, or improving an infection, disease, or condition caused by Streptococcus pneumoniae in a subject, which includes administering an immunologically effective amount of the immunogenic composition of the present description to the subject. In some exemplary embodiments, the disease or infectious condition is selected from a group consisting of pneumonia, paranasal sinusitis, otitis media, acute otitis media, meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, myelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and pyocephaly. In one aspect, the present description provides a method for inducing an immune response to Streptococcus pneumoniae in a subject, which includes administering an immunologically effective amount of the immunogenic composition of the present description to the subject. In one exemplary embodiment, the immunogenic composition described herein is used as a vaccine. In such an exemplary embodiment, the immunogenic composition described herein can be used to prevent Streptococcus infection. IF-2019-79554467-APN-ANP#INPI Page 47 of 67 113 pneumoniae in a subject. Accordingly, in one aspect, the present description provides a method for preventing Streptococcus pneumoniae infection in a subject, which includes administering an immunologically effective amount of the immunogenic composition of the present description to the subject. In some of these exemplary embodiments, the infection is selected from a group consisting of pneumonia, paranasal sinusitis, otitis media, acute otitis media, meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, myelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and pyocephaly. In one exemplary embodiment, the vaccinated subject is a mammal, for example, a human, cat, sheep, pig, horse, bovine, or dog. In one exemplary embodiment, the immunogenic composition described herein is for use in a method for preventing, treating, or ameliorating an infection, disease, or condition associated with Streptococcus pneumoniae in a subject. In some of these exemplary embodiments, the infection, disease, or condition is selected from a group consisting of pneumonia, paranasal sinusitis, otitis media, acute otitis media, meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, myelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and pyocephaly. In one exemplary embodiment, the immunogenic composition described herein is for use as a vaccine. In such an exemplary embodiment, the immunogenic composition described herein can be used to prevent Streptococcus infection. IF-2019-79554467-APN-ANP#INPI Page 48 of 67 114 pneumoniae in a subject. Accordingly, in one exemplary embodiment, the immunogenic composition described herein is for use in a method for preventing Streptococcus pneumoniae infection in a subject. In some of these exemplary embodiments, the infection is selected from a group consisting of pneumonia, paranasal sinusitis, otitis media, acute otitis media, meningitis, bacteremia, septicemia, pyothorax, conjunctivitis, myelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and pyocephaly. In one exemplary embodiment, the vaccinated subject is a mammal, for example, a human, cat, sheep, pig, horse, bovine, or dog. The immunogenic composition described herein can be used to protect against or treat a human susceptible to pneumococcal infection by administering the immunogenic compositions via a systemic or mucosal route. In one exemplary embodiment, the immunogenic composition described herein is administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In ELISA (enzyme-linked immunosorbent assay), antibodies from the sera of vaccinated subjects are incubated with polysaccharides that have been adsorbed onto a solid support. The bound antibodies are detected using enzyme-conjugated secondary detection antibodies. IF-2019-79554467-APN-ANP#INPI Page 49 of 67 115 The ELISA measures IgG-specific capsular polysaccharide (PS) antibodies against Streptococcus pneumoniae present in human serum. When dilutions of human sera are added to microtiter plates coated with specific capsular PS, antibodies specific to that capsular PS bind to the plates. The plate-bound antibodies are detected using an alkaline phosphatase-labeled goat IgG anti-human antibody followed by a p-nitrophenyl phosphate substrate. The optical density of the colored final product is proportional to the amount of anticapsular PS antibody present in the serum. In an exemplary embodiment, the immunogenic composition containing one or more saccharide-protein conjugates of Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 can elicit IgG antibodies in humans that are capable of binding to the polysaccharide of Streptococcus pneumoniae serotype 15B at a concentration of at least 0.05, 0.1, 0.2, 0.3, 0.35, 0.4, or 0.5 μg / mL as determined by the ELISA assay. In an exemplary embodiment, the immunogenic composition containing one or more saccharide-protein conjugates selected from Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20, when administered to a subject, can induce the formation of antibodies capable of killing Streptococcus pneumoniae of one or more selected serotypes of Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 by opsonophagocytosis. In an exemplary embodiment, the immunogenic composition containing one or more saccharide-protein conjugates selected from Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20, when tested in a IF-2019-79554467-APN-ANP#INPI Page 50 of 67 116 OPA assay, has a higher OPA titer than the OPA titer obtained with a non-conjugated native capsular polysaccharide of Streptococcus pneumoniae. The pneumococcal opsonophagocytic assay (OPA), which measures the destruction of Streptococcus pneumoniae cells by phagocytic effect in the presence of functional antibodies and complement, is considered an important surrogate for evaluating the effectiveness of pneumococcal vaccines. The opsonophagocytic assay (OPA) can be performed by incubating together a mixture of Streptococcus pneumoniae cells, a heat-inactivated human serum for testing, differentiated HL-60 cells (phagocytes), and an exogenous complement source (e.g., baby rabbit complement). Opsonophagocytosis occurs during incubation, and bacterial cells coated with antibody and complement are destroyed by this process. The colony-forming units (CFUs) of surviving bacteria that escape opsonophagocytosis are determined by plating the assay mixture. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial count in the control wells without the test serum. The OPA titer is interpolated from the two dilutions that encompass this 50% destruction limit. A stock split of 1:8 or higher is considered a positive outcome in this type of takeover bid. In an exemplary embodiment, the immunogenic composition containing one or more saccharide-protein conjugates selected from Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 is capable of inducing a titer of at least 1:8 against one or more selected serotypes of IF-2019-79554467-APN-ANP#INPI Page 51 of 67 117 Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 in at least 50% of subjects, as determined by the opsonophagocytic destruction assay (OPA). In an exemplary embodiment, the immunogenic composition containing one or more saccharide-protein conjugates selected from Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 is capable of inducing a titer of at least 1:8 against Streptococcus pneumoniae serotypes 2, 9N, 17F, and 20 in at least 60%, 70%, 80%, 90%, or 93% of subjects, as determined by the opsonophagocytic destruction assay (OPA). 7. Subject to treatment with the immunogenic composition of the present description As described in this document, the immunogenic composition described herein can be used in various therapeutic or prophylactic methods to prevent, treat, or improve a bacterial infection, disease, or condition in a subject. In one specific exemplary form of realization, the subject is a human being. In a more specific exemplary form of realization, the subject is a newborn (i.e., less than three months old), an infant (i.e., from three months to one year old), or a toddler (i.e., from one to four years old). In one exemplary embodiment, the immunogenic composition described herein is for use as a vaccine. In this exemplary embodiment, the subject to be vaccinated may be under 1 year of age. For example, the subject to be vaccinated may be approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, IF-2019-79554467-APN-ANP#INPI Page 52 of 67 118 approximately 8, approximately 9, approximately 10, approximately 11, or approximately 12 months of age. In one exemplary embodiment, the subject to be vaccinated is approximately 2, approximately 4, or approximately 6 months of age. In another exemplary embodiment, the subject to be vaccinated is under 2 years of age. For example, the subject to be vaccinated may be between 12 and 15 months of age. In some cases, only one dose of the immunogenic composition as described herein is required. However, in some circumstances, a second, third, or fourth dose may be administered. In an exemplary form of realization of the present description, the subject to be vaccinated is a human adult 50 years of age or older, more specifically a human adult 55 years of age or older. In an exemplary form of implementation, the subject to be vaccinated is a human adult aged 65 years or older, 70 years or older, 75 years or older, or 80 years or older. In an exemplary scenario, the subject to be vaccinated is an immunocompromised individual, specifically a human being. An immunocompromised individual is generally defined as a person who exhibits an attenuated or reduced capacity to mount a normal humoral or cellular defense against the challenge of infectious agents. In an exemplary realization of the present description, the immunocompromised subject to be vaccinated suffers from a disease or condition that affects the immune system and results in an antibody response that is insufficient to protect against or treat the disease. IF-2019-79554467-APN-ANP#INPI Page 53 of 67 119 In one exemplary form, the disease is a primary immunodeficiency disorder. Specifically, the primary immunodeficiency disorder is selected from a group consisting of combined T- and B-cell immunodeficiencies, antibody deficiencies, well-defined syndromes, immune dysregulation diseases, phagocyte disorders, innate immunity deficiencies, autoinflammatory disorders, and complement deficiencies. In one specific exemplary embodiment of the present description, the immunocompromised subject to be vaccinated suffers from a selected disease from a group consisting of: HIV infection, acquired immunodeficiency syndrome (AIDS), cancer, chronic heart or lung disorders, congestive heart failure, diabetes mellitus, chronic liver disease, alcoholism, cirrhosis, cerebrospinal fluid leaks, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), spleen dysfunction (e.g., sickle cell disease), lack of spleen function (asplenia), blood cancer, leukemia, multiple myeloma, Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome, and asthma. In an exemplary realization of the present description, the immunocompromised subject to be vaccinated suffers from malnutrition. In one specific exemplary embodiment of the present description, the immunocompromised subject to be vaccinated is taking a medication or treatment that decreases the body's resistance to infection. In a specific exemplary realization of the present description, the immunocompromised subject to be vaccinated is a smoker. IF-2019-79554467-APN-ANP#INPI Page 54 of 67 120 In one exemplary embodiment specific to the present description, the immunocompromised subject to be vaccinated has a white blood cell count (leukocyte count) below 5 x 109 cells / l, or below 4 x 109 cells / l, or below 3 x 109 cells / L, or below 2 x 109 cells / L, or below 1 x 109 cells / L, or below 0.5 x 109 cells / L, or below 0.3 x 109 cells / L, or below 0.1 x 109 cells / L. The white blood cell count (leukocyte count) refers to the number of white blood cells (WBCs) in the blood. WBCs are usually measured as part of the CBC (complete blood count). White blood cells are the cells that fight infection in the blood and are distinct from red blood cells (which carry oxygen), known as erythrocytes. There are different types of white blood cells, including neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T lymphocytes (T cells), B lymphocytes (B cells), monocytes, eosinophils, and basophils. All types of white blood cells are reflected in the white blood cell count. The normal range for a white blood cell count is usually between 4,300 and 10,800 cells per cubic millimeter of blood. This can also be called the leukocyte count and can be expressed in the international unit as 4.3–10.8 × 10⁹ cells / L. In one specific exemplary embodiment of the present description, the immunocompromised subject to be vaccinated suffers from neutropenia. In one specific exemplary embodiment of the present description, the immunocompromised subject to be vaccinated has a neutrophil count below 2 x 10⁹ cells / L, or below 1 x 10⁹ cells / L, or below 0.5 x 10⁹ cells / L, or below 0.1 x 10⁹ cells / L, or less than 0.05 x 10⁹ cells / L. IF-2019-79554467-APN-ANP#INPI Page 55 of 67 121 A 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 helps prevent and fight infections. The most common reason cancer patients experience neutropenia is as a side effect of chemotherapy. Chemotherapy-induced neutropenia increases a patient's risk of infection and can disrupt cancer treatment. In one specific exemplary embodiment of the present description, the immunocompromised subject to be vaccinated has a CD4+ cell count below 500 / mm3, a CD4+ cell count below 300 / mm3, a CD4+ cell count below 200 / mm3, a CD4+ cell count below 100 / mm3, a CD4+ cell count below 75 / mm3, or a CD4+ cell count below 50 / mm3. CD4 cell test results are typically reported as the number of cells per mm3. Normal CD4 counts range from 500 to 1600, and CD8 counts range from 375 to 1100. CD4 counts decrease dramatically in people with HIV. In an exemplary form of realization of the present description, any of the immunocompromised subjects described herein is a human male or a human female. 8. Regime In some cases, only one dose of the immunogenic composition as described herein is required, but in certain circumstances, such as conditions of severe immunodeficiency, a second, third, or fourth dose may be administered after vaccination. IF-2019-79554467-APN-ANP#INPI Page 56 of 67 122 Initial, subjects may receive one or more booster immunizations at appropriate intervals. In one exemplary embodiment, the vaccination program for the immunogenic composition described herein is a single-dose program. In one specific exemplary embodiment, the single-dose program is for a healthy person who is at least 2 years of age. In one exemplary embodiment, the vaccination schedule for the immunogenic composition described herein is a multi-dose schedule. In one specific exemplary embodiment, the multi-dose schedule consists of a series of two doses separated by an interval of approximately one to two months. In one specific exemplary embodiment, the multi-dose schedule consists of a series of two doses separated by an interval of approximately one month, or a series of two doses separated by an interval of approximately two months. In another exemplary embodiment, the multiple-dose program consists of a series of 3 doses separated by an interval of approximately 1-2 months. In another exemplary embodiment, the multiple-dose program consists of a series of 3 doses separated by an interval of approximately 1 month, or a series of 3 doses separated by an interval of approximately 2 months. In another exemplary embodiment, the multiple-dose program consists of a series of three doses separated by an interval of approximately one to two months, followed by a fourth dose approximately 10 to 13 months after the first dose. In another exemplary embodiment, the program IF-2019-79554467-APN-ANP#INPI Page 57 of 67 123 multiple dose consists of a series of 3 doses separated by an interval of approximately 1 month followed by a fourth dose approximately 10-13 months after the first dose, or a series of 3 doses separated by an interval of approximately 2 months followed by a fourth dose approximately 10-13 months after the first dose. In one exemplary implementation, the multiple-dose program consists of at least one dose (e.g., 1, 2, or 3 doses) in the first year of age, followed by at least one dose for young children. In one exemplary embodiment, the multiple-dose schedule consists of a series of 2 or 3 doses separated by an interval of approximately 1–2 months (e.g., 28–56 days between doses), beginning at 2 months of age, and followed by a toddler dose at 12–18 months of age. In another exemplary embodiment, the multiple-dose schedule consists of a series of 2 doses separated by an interval of approximately 2 months, beginning at 2 months of age and followed by a toddler dose at 12–15 months of age. In an exemplary implementation, the multiple-dose program consists of a series of 4 vaccine doses at 2, 4, 6, and 12-15 months of age. In one exemplary embodiment, the first dose is administered on day 0 and one or more additional doses are administered at intervals that vary from IF-2019-79554467-APN-ANP#INPI Page 58 of 67 124 approximately 2 to approximately 24 weeks, specifically with a dosing interval of 4-8 weeks. In an exemplary implementation, the first dose is administered on day 0 and the additional dose is administered approximately 3 months later. The immunogenic conjugate described herein can be used as an effective immunogenic conjugate because it exhibits superior stability and high conjugation yield. Advantageous effects The present description provides a saccharidoprotein conjugate vaccine capable of providing an improved antibody titer compared to a saccharide vaccine. This description may provide a higher antibody titer. The pneumococcal vaccine described here has fewer side effects. The vaccine described here can be given to infants, children, and the elderly. MODE FOR DESCRIPTION The following examples illustrate this description in more detail. However, the examples in this description may be modified in various other ways, and the description should be interpreted as being limited by the examples provided. The examples in this description are provided to aid in understanding it. IF-2019-79554467-APN-ANP#INPI Page 59 of 67 125 Example 1. Preparation of saccharide-protein conjugate from Streptococcus pneumoniae serotype 9N Cell bank preparation Streptococcus pneumoniae serotype 9N (ATCC 6309) was acquired from the American Type Culture Collection (ATCC). For strain proliferation and elimination of animal-derived components, the seed stock was cultured for several generations. The stock vial was kept in a refrigerator (< -70°C) with synthetic glycerol as a cryoprotectant. For cell bank preparation, the cell culture was grown on a soy-based medium. Before freezing, the cells were concentrated by centrifugation, and after removing the used medium, the cell pellets were resuspended in fresh medium containing a cryoprotectant (e.g., synthetic glycerol). Fermentation Cell bank cultures were inoculated into culture bottles containing a soy-based medium. Until growth conditions were met, the culture was incubated at a constant temperature without agitation. The culture was then inoculated into a seed fermenter containing soy-based medium, with controlled temperature, pH, and agitation speed, using a culture bottle. Fermentation was terminated after growth ceased or the fermenter reached its working capacity. After fermentation was completed by adding a deactivator, cell debris was removed using a combination of continuous-flow centrifugation and filtration. Purification The pneumococcal polysaccharide purification process consisted of IF-2019-79554467-APN-ANP#INPI Page 60 of 67 126 multilayer filtration, repeated concentration / diafiltration and filtration / elution. Activation The final polysaccharide concentration was adjusted to approximately 2.0 g / L by sequentially adding WFI in a calculated amount. If necessary, the reaction pH was adjusted to approximately 6.0. After pH adjustment, the reaction temperature was set to 21–25°C. Approximately 0.024–0.189 mg of sodium periodate per 1 mg of sugar was added to initiate the oxidation. The oxidation reaction was carried out for 16–20 hours at 21–25°C. The activated polysaccharide was concentrated and diafiltered using a 100-kDa MWCO ultrafiltration membrane. Diafiltration was performed at a water filtration index (WFI) of 10 times the diafiltration volume. The purified activated polysaccharide was then stored at 2–8°C. The purified activated polysaccharide was characterized by (i) saccharide concentration determined by colorimetric assay, (ii) aldehyde concentration determined by colorimetric assay, (iii) degree of oxidation, and (iv) molecular weight measured by SEC-MALLS. SEC-MALLS is used to determine the molecular weight of polysaccharides and polysaccharide-protein conjugates. SEC is used to separate the polysaccharide based on hydrodynamic volume. A refractive index (RI) detector and a multi-angle laser light scattering (MALLS) detector are used to determine the molecular weight. When light reacts with a material, it is scattered. The amount of scattered light is related to the concentration, the square of the refractive index change (dn / dc), and the molar mass of the material. The molecular weight is calculated based on the light scattered by the MALLS detector and the concentration signal from the RI detector. The degree of oxidation (DO) of the activated polysaccharide is determined as the IF-2019-79554467-APN-ANP#INPI Page 61 of 67 127 moles of sugar repeating units divided by the moles of aldehyde. The moles of sugar repeating units are determined using various colorimetric techniques, for example, using an anthrone assay. And, the moles of aldehyde are determined using the ParkJohnson colorimetric assay. Specifically, the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide obtained using the method described above has an oxidation degree of 2-19 and a molecular weight of approximately 200-700 kDa. Conjugation The activated polysaccharide was combined with the carrier protein CRM197, in a ratio of 0.5–2 g of CRM197 per 1 g of activated polysaccharide. The combined mixture was then lyophilized. The lyophilized mixture of activated polysaccharide and CRM197 was stored at -20°C. The lyophilized mixture of the activated polysaccharide and CRM197 was reconstituted in a 0.1 M sodium phosphate solution and thoroughly mixed. The final concentration of the polysaccharide in the reaction solution was approximately 10–20 g / L. After initiating the conjugation by adding 1.0–1.2 molar equivalents of sodium cyanoborohydride (NaBH3CN) to the reaction mixture, the reaction was carried out at 35–39°C for 44–52 hours. The conjugation reaction was terminated by adding a 0.9% sodium chloride solution of the same volume as the conjugation reaction solution, followed by the addition of 1.8–2.2 molar equivalents of sodium borohydride (NaBH4) to protect the unreacted aldehyde. The protection reaction was carried out for 3–6 hours at 21–25°C. IF-2019-79554467-APN-ANP#INPI Page 62 of 67 128 The conjugate solution was diluted with 0.9% sodium chloride solution for concentration and diafiltration using a 100 kDa MWCO membrane. The diluted conjugate solution was filtered through a 0.8–0.45 μm filter and purified by concentration and diafiltration. Diafiltration using a 100 kDa MWCO membrane was performed using 0.9% sodium chloride solution in 15–40 times the diafiltration volume. After diafiltration was complete, the remaining solution was filtered through a 0.2 µm filter. The conjugate solution was diluted to a concentration below approximately 0.55 mg / mL, sterilized by filtration, and then stored at 28°C. The purified 9N serotype conjugate was characterized, in particular, by (i) the protein concentration determined by colorimetric assay (Lowry), (ii) the aldehyde concentration determined by colorimetric assay, (iii) the saccharide-protein ratio, (iv) the molecular size distribution determined by size exclusion chromatography (CL-4B), and (v) the molecular weight measured by SEC-MALLS. The change in the characteristics of the 9N serotype conjugate was observed while varying the oxidation state (OD). The results are summarized in Table 1. Table 1 Number of conjugates 1 2 3 4 5 6 Molecular weight of activated polysaccharide, kDa 582 619 459 563 490 427 IF-2019-79554467-APN-ANP#INPI Page 63 of 67 129 Number of conjugates 1 2 3 4 5 6 OD 18.2 9.4 7.4 6.7 4.3 2.3 Input ratio (P:S) 0.8:1 Polysaccharide concentration in conjugation reaction solution, g / L 20.0 % conjugate yield 53 43 39 32 33 39 Saccharide-protein ratio 2.1 1.5 1.3 1.1 1.0 0.78 % free saccharide 44 28 22 20 21 31 % molecular weight distribution 52 49 50 55 44 31 Molecular weight of conjugate, kDa 860 1110 1912 1168 1189 1160 The change in the characteristics of the 9N serotype conjugate was observed while varying the ratio of the activated polysaccharide and CRM197 combination during lyophilization. The results are summarized in Table 2. Table 2 Number of conjugates 7 8 9 10 11 Molecular weight of activated polysaccharide, kDa 287 OD 5.6 IF-2019-79554467-APN-ANP#INPI Page 64 of 67 130 Input ratio (P:S) 2:1 1.5:1 1:1 0.67:1 0.5:1 Polysaccharide concentration in conjugation reaction solution, g / L 20.0 % conjugate yield 25 50 43 41 66 Saccharide-protein ratio 0.71 0.85 1.0 1.2 1.8 % free saccharide 5 6 15 27 62 % molecular weight distribution 52 58 50 40 22 Molecular weight of conjugate, kDa 3,720 3,713 1,327 1,016 545 The change in the characteristics of the 9N serotype conjugate was observed while varying the concentration of the polysaccharide in the conjugation reaction solution. The results are summarized in Table 3. Table 3 Number of conjugates 12 13 14 15 16 Molecular weight of activated polysaccharide, kDa 560 OD 6.1 Input ratio (P:S) 0.8:1 Concentration of polysaccharide in conjugation reaction solution, 10.0 12.5 15.0 17.5 20.0 IF-2019-79554467-APN-ANP#INPI Page 65 of 67 131 g / L % yield of conjugate 20 31 28 40 42 Saccharide-protein ratio 1.0 1.0 0.93 0.99 0.97 % free saccharide 32 30 22 21 18 % molecular weight distribution 17 27 40 47 54 Molecular weight of conjugate, kDa 560 546 845 932 1.438 Example 2. Immunogenicity Analysis A monovalent conjugate composition was formulated containing the saccharide-protein conjugate of Streptococcus pneumoniae serotype 9N with CRM197. The immunogenicity of the immunogenic monovalent compositions in Tables 1-3 was analyzed in rabbits by ELISA. The serum concentration of serotype-specific IgG was determined. Five female New Zealand White rabbits weighing 2.5–3.5 kg were immunized with the proposed human clinical dose (2.2 μg conjugate + 0.25 mg / mL aluminum as AlPO4) at week 0 via intramuscular injection. The rabbits were re-immunized at week 2 with the conjugate vaccine at the same dose, and blood samples were collected at week 4. A serotype-specific ELISA was performed on serum samples at week 0 and week 4. The results of the analysis are shown in Table 4. Rabbits immunized with the monovalent conjugate composition (conjugate number 8) showed IF-2019-79554467-APN-ANP#INPI Page 66 of 67 132 a significant increase in total IgG titer for serotype 9N. Rabbits immunized with other conjugates also showed a significant increase in total IgG titer. Table 4 shows the result of the IgG concentration measurement after immunizing rabbits with conjugate number 8 from Table 2. Table 4 IgG Concentration (U / ml) Serotype Pre-immunization Post-immunization 9N 130.0 656,345.3 IF-2019-79554467-APN-ANP#INPI Page 67 of 67 Argentine Republic - National Executive Branch 2019 - Year of Exports Additional Signature Sheet Graphic Report Number: IF-2019-795 54467-APN-ANP#INPI CITY OF BUENOS AIRES Tuesday, September 3, 2019 Reference: 20190100274 The document was imported by the GEDO system with a total of 67 page(s). Digitally signed by GESTION DOCUMENTAL ELECTRONICA - GDE DN: cn=ELECTRONIC DOCUMENTARY MANAGEMENT - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.09.03 06:35:39 -03'00' German Sapia Administrative Assistant National Patent Administration National Institute of Industrial Property Digitally signed by GESTION DOCUMENTAL ELECTRONICA GDE DN: cn=GEST10N ELECTRONIC DOCUMENTARY - GDE, c=AR, o=GOVERNMENT SECRETARY OF MODERNIZATION, ou=ADMINISTRATIVE MODERNIZATION SECRETARY, serialNumber=CUIT 30715117564 Date: 2019.09.03 06:35:40 -03'00'

Claims

1. A multivalent pneumococcal conjugate composition with a mixed carrier, characterized in that said composition comprises 21 different pneumococcal protein-capsular polysaccharide conjugates and a physiologically acceptable vehicle, wherein each pneumococcal protein-capsular polysaccharide conjugate comprises a carrier protein conjugated to a capsular polysaccharide of a different Streptococcus pneumoniae serotype, wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, wherein the carrier protein is CRM197 or tetanus toxoid, and wherein four of the polysaccharides capsular polysaccharides are conjugated with tetanus toxoid and the remaining capsular polysaccharides are conjugated with CRM197, wherein the four capsular polysaccharides that are conjugated with tetanus toxoid are serotypes 15B, 22F and two serotypes selected from the group consisting of serotypes 1, 3 and 5.The following 20 demands follow.