Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Abstract

To provide an effective vaccine against Streptococcus pneumoniae infection, which can be safely produced in a large amount.SOLUTION: The present invention relates to novel immunogenic compositions comprising conjugated Streptococcuspneumoniae capsular saccharide antigens (glycoconjugates), kits comprising said immunogenic compositions and uses thereof. The invention also relates to the vaccination of human subjects, in particular infants and elderly, against pneumococcal infection using said novel immunogenic compositions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of immunogenic compositions and vaccines, their manufacture and the use of such compositions in medicine.

[0002] More particularly, the present invention relates to isolated Streptococcus pneumoniae serotype 12F saccharides, glycoconjugates thereof, methods for making Streptococcus pneumoniae serotype 12F glycoconjugates, and immunogenic compositions comprising Streptococcus pneumoniae serotype 12F glycoconjugates.

[0003] The present invention also relates to analytical methods for analyzing isolated S. pneumoniae serotype 12F polysaccharide, reduced serotype 12F polysaccharide, or Streptococcus pneumoniae serotype 12F glycoconjugates.

[0004] The Streptococcus pneumoniae serotype 12F saccharides and glycoconjugates of the present invention may be used as vaccines. [Background technology]

[0005] Infections caused by Streptococcus pneumoniae are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, while spread of bacteria within the respiratory tract can lead to middle ear infections, sinusitis, or recurrent bronchitis. Compared to invasive disease, noninvasive manifestations are usually less severe but occur significantly more frequently.

[0006] In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, affecting an estimated 100 adults per 100,000 annually. The corresponding figures for febrile bacteremia and meningitis are 15–19 per 100,000 and 1–2 per 100,000, respectively. The risk of developing one or more of these conditions is much higher in infants and the elderly, as well as immunocompromised individuals of any age. Even in economically developed regions, invasive pneumococcal disease has a high mortality rate. While the average mortality rate for adults with pneumococcal pneumonia is 10–20%, it can exceed 50% in high-risk groups. Pneumonia is by far the most common cause of pneumococcal death worldwide.

[0007] Streptococcus pneumoniae (pneumococcus), the etiological agent of pneumococcal disease, is a Gram-positive, encapsulated cocci surrounded by a polysaccharide capsule. Differences in the composition of this capsule allow serological differentiation among approximately 91 capsular types, some of which are frequently associated with pneumococcal disease and others that are rarely associated. Invasive pneumococcal infections include pneumonia, meningitis, and febrile bacteremia; common noninvasive manifestations are otitis media, sinusitis, and bronchitis, among others.

[0008] T-independent antigens, such as saccharides, are antigens that induce antibody production through B lymphocytes without the involvement of T cells. Conjugation of T-independent antigens to carrier proteins has been established as a way to allow T cell help to be part of the immune response to normally T-independent antigens. Successful conjugate vaccines have been developed by conjugating bacterial capsular saccharides to carrier proteins (carrier proteins with known efficacy in converting T-independent saccharide antigens into T-dependent antigens that can trigger immune memory responses). Several carrier proteins are known in the art, including tetanus toxoid, diphtheria toxoid, CRM 197, and protein D from Haemophilus influenzae are used as carrier proteins in commercial vaccines. Pneumococcal conjugate vaccines (PCVs) are pneumococcal vaccines used to protect against disease caused by S. pneumoniae (pneumococcus). Three PCV vaccines are currently available on the global market: PREVNAR® (PREVENAR® in some countries) (7-valent vaccine), SYNFLORIX® (10-valent vaccine), and PREVNAR13® (PREVENAR13® in some countries) (13-valent vaccine). Summary of the Invention [Problem to be solved by the invention]

[0009] There remains a great need for an effective vaccine against Streptococcus pneumoniae infections that is safe and can be produced in large quantities. [Means for solving the problem]

[0010] To meet these and other needs, the present invention relates to an isolated polysaccharide having the following repeating units:

[0011] [ka]

[0012] where n represents the number of repeating units and X represents either N-acetylgalactosamine or 4-keto-N-acetyl-quinovosamine (2-acetamido-2,6-dideoxy-xylo-hexos-4-ose).

[0013] In one embodiment, the isolated polysaccharide comprises from about 99.9 to about 50 N-acetylgalactosamine residues and from about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0014] In one aspect, the invention relates to an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 50 N-acetylgalactosamine residues and from about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0015] The present invention further relates to a S. pneumoniae serotype 12F glycoconjugate prepared by a process comprising the steps of: a) reacting the isolated polysaccharide with an activating agent to produce an activated sugar; and b) reacting the activated sugar with a carrier protein.

[0016] In another aspect, the invention relates to a Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 saccharide repeating units of the polysaccharide. In a specific aspect, the invention further relates to a Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0017] In one aspect, the glycoconjugates are prepared using reductive amination.

[0018] The invention further relates to immunogenic compositions comprising said polysaccharides or glycoconjugates and to their use as pharmaceuticals, in particular as vaccines. [Brief explanation of the drawings]

[0019] [Figure 1A]Schematic diagram showing the organization and population of the pneumococcal polysaccharide 12F repeat unit, which contains a primary population (A) at approximately 75-80 mol%, consistent with Leontein et al. ((1981) Can. J. Chem. 59:2081-2085), and a secondary population (B) at approximately 20-25 mol%, characterized by the replacement of GalNAc with Sug (a keto sugar). In the secondary spin system, the shaded circles indicate site-specific 13C and / or 1H resonances of main-chain and branching residues that are significantly affected by the incorporation of Sug residues. [Figure 1B] Schematic diagram showing the organization and population of the pneumococcal polysaccharide 12F repeat unit, which contains a primary population (A) at approximately 75-80 mol%, consistent with Leontein et al. ((1981) Can. J. Chem. 59:2081-2085), and a secondary population (B) at approximately 20-25 mol%, characterized by the replacement of GalNAc with Sug (a keto sugar). In the secondary spin system, the shaded circles indicate site-specific 13C and / or 1H resonances of main-chain and branching residues that are significantly affected by the incorporation of Sug residues. [Figure 1C] Schematic representation of an average pneumococcal polysaccharide 12F repeat unit, with the central backbone residue shown as either GalNAc or Sug based on a statistical average (75% / 25%). [Figure 2] Figure 1 shows the assignment of 1H and 13C chemical shifts of the hydrated form of pneumococcal polysaccharide 12F Sug residue. Sug (also referred to herein as 4-keto-N-acetyl-quinovosamine, 2-acetamido-2,6-dideoxy-D-xylo-4-hexulose, or 4KQ) is incorporated into the backbone of the repeating unit by replacing a GalNAc residue. [Figure 3] FIG. 1 shows the ketone / hydrate equilibrium. [Figure 4] FIG. 11 shows 11 NOESY correlations providing further support that D-Sug replaces GalNAc in the polysaccharide 12F repeat unit. [Figure 5] FIG. 1 shows the GalNAc-containing repeating units of the 12F polysaccharide as detected by in-source collision-induced dissociation (IS-CID). [Figure 6] FIG. 1 is a schematic diagram of serotype 12F Sug residue (4-keto-N-acetyl-quinovosamine) showing the ketone / hydrate equilibrium in aqueous media, as well as the changes resulting from specific ketone reduction using NaBH 4 . [Figure 7] FIG. 1 is a schematic diagram showing serotype 12F repeat units containing Sug residues after reduction with NaBD4. [Figure 8] Immune sera from subjects immunized with a multivalent vaccine containing the 12F conjugate (12F conj.), a multivalent vaccine containing plain 12F (12F plain), or a multivalent vaccine that did not contain 12F polysaccharide were tested for their ability to elicit bacterial killing responses of isolates with 4KQ modification levels ranging from 1.9% to 27.5%. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based, in part, on the identification of a novel pneumococcal polysaccharide structure through the use of NMR spectroscopy. The structure provided herein is believed to be the first identification or first correct identification of S. pneumoniae serotype 12F.

[0021] S. pneumoniae serotype 12F polysaccharide was produced and purified from various strains. The produced (and purified) polysaccharide was used to prepare polysaccharide-protein conjugates (glycoconjugates). S. pneumoniae serotype 12F has a unique polysaccharide structure, which leads to a unique conjugate production process.

[0022] 1. The isolated Streptococcus pneumoniae serotype 12F glycoprotein of the present invention As used herein, the term "isolated" in reference to polysaccharides refers to the isolation of S. pneumoniae serotype-specific capsular polysaccharides from purified polysaccharides using purification techniques known in the art, including the use of centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography. Generally, isolated polysaccharides refer to the partial removal of proteins, nucleic acids, and nonspecific endogenous polysaccharides (C-polysaccharides). Isolated polysaccharides contain less than 10%, 8%, 6%, 4%, or 2% protein impurities and / or nucleic acids. Isolated polysaccharides contain less than 20% C-polysaccharides relative to the type-specific polysaccharides.

[0023] The structure of the S. pneumoniae serotype 12F capsular polysaccharide has been previously published (Leontein et al. (1981) Can. J. Chem. 59:2081-2085).

[0024] According to Leontein et al., the polysaccharide repeating unit of serotype 12F consists of a linear trisaccharide backbone (one N-acetylfucosamine (FucpNAc), one N-acetylgalactosamine (GalpNAc), and one N-acetylmannuronic acid (ManpNAcA)) with two branches: a pendant α-galactopyranose (Galp) linked at C3 of FucpNAc and an α-Glcp-(1→2)-α-Glcp disaccharide branch linked at C3 of ManpNAcA.

[0025] The present inventors have surprisingly discovered that the structure of S. pneumoniae serotype 12F is different: for the first time, the inventors have found that the serotype 12F polysaccharide indeed contains a partial replacement of N-acetyl-galactosamine with 4-keto-N-acetyl-quinovosamine (also referred to herein as Sug, D-Sug, 2-acetamido-2,6-dideoxy-D-xylo-4-hexulose, or 4KQ).

[0026] Various clinical serotype 12F isolates and various serotype 12F strains were examined for the presence of 4KQ incorporation. All clinical isolates studied had some level of 4KQ incorporation, indicating that 4KQ substitution is common in clinical serotype 12F isolates.

[0027] Thus, in one embodiment, the present invention provides an isolated polysaccharide having the following repeating unit:

[0028] [ka]

[0029] where n represents the number of repeating units and X represents either N-acetylgalactosamine or 4-keto-N-acetyl-quinovosamine. In one embodiment, the present invention provides an isolated polysaccharide having the following repeating unit:

[0030] [ka]

[0031] wherein n represents the number of repeating units, X represents either N-acetylgalactosamine or 4-keto-N-acetyl-quinovosamine, and the polysaccharide contains from about 99.9 to about 50 N-acetylgalactosamine residues and from about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0032] In certain embodiments, the isolated polysaccharide comprises from about 99.1 to about 50 N-acetylgalactosamine residues and from about 0.9 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0033] In certain embodiments, the isolated polysaccharide comprises about 99 to about 50 N-acetylgalactosamine residues and about 1 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0034] In certain embodiments, the isolated polysaccharide comprises about 98 to about 50 N-acetylgalactosamine residues and about 2 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 97 to about 50 N-acetylgalactosamine residues and about 3 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 96 to about 50 N-acetylgalactosamine residues and about 4 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 95 to about 50 N-acetylgalactosamine residues and about 5 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 94 to about 50 N-acetylgalactosamine residues and about 6 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 93 to about 50 N-acetylgalactosamine residues and about 7 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 92 to about 50 N-acetylgalactosamine residues and about 8 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 91 to about 50 N-acetylgalactosamine residues and about 9 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 90 to about 50 N-acetylgalactosamine residues and about 10 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0035] In certain embodiments, the isolated polysaccharide comprises from about 99.1 to about 55 N-acetylgalactosamine residues and from about 0.9 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0036] In certain embodiments, the isolated polysaccharide comprises from about 99 to about 55 N-acetylgalactosamine residues and from about 1 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0037] In certain embodiments, the isolated polysaccharide comprises about 98 to about 55 N-acetylgalactosamine residues and about 2 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 97 to about 55 N-acetylgalactosamine residues and about 3 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 96 to about 55 N-acetylgalactosamine residues and about 4 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 95 to about 55 N-acetylgalactosamine residues and about 5 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 94 to about 55 N-acetylgalactosamine residues and about 4 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 93 to about 55 N-acetylgalactosamine residues and about 7 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 92 to about 55 N-acetylgalactosamine residues and about 8 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 91 to about 55 N-acetylgalactosamine residues and about 9 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 90 to about 55 N-acetylgalactosamine residues and about 10 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0038] In certain embodiments, the isolated polysaccharide comprises from about 99.9 to about 75 N-acetylgalactosamine residues and from about 0.1 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0039] In certain embodiments, the isolated polysaccharide comprises from about 99.1 to about 75 N-acetylgalactosamine residues and from about 0.9 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0040] In certain embodiments, the isolated polysaccharide comprises from about 99 to about 75 N-acetylgalactosamine residues and from about 1 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0041] In certain embodiments, the isolated polysaccharide comprises about 98 to about 75 N-acetylgalactosamine residues and about 2 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 97 to about 75 N-acetylgalactosamine residues and about 3 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 96 to about 75 N-acetylgalactosamine residues and about 4 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 95 to about 75 N-acetylgalactosamine residues and about 5 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 94 to about 75 N-acetylgalactosamine residues and about 4 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 93 to about 75 N-acetylgalactosamine residues and about 7 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 92 to about 75 N-acetylgalactosamine residues and about 8 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 91 to about 75 N-acetylgalactosamine residues and about 9 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide. In certain embodiments, the isolated polysaccharide comprises about 90 to about 75 N-acetylgalactosamine residues and about 10 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0042] In certain embodiments, the isolated polysaccharide comprises about 99.9 N-acetylgalactosamine residues and about 0.1 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0043] In certain embodiments, the isolated polysaccharide comprises about 99.8 N-acetylgalactosamine residues and about 0.2 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0044] In certain embodiments, the isolated polysaccharide comprises about 99.5 N-acetylgalactosamine residues and about 0.5 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeating units of the polysaccharide.

[0045] In certain embodiments, the isolated polysaccharide comprises about 99 N-acetylgalactosamine residues and about 1 4-keto-N-acetyl-quinovosamine residue for every 100 saccharide repeat units of the polysaccharide.

[0046] In certain embodiments, the isolated polysaccharide comprises about 98 N-acetylgalactosamine residues and about 2 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0047] In certain embodiments, the isolated polysaccharide comprises about 97 N-acetylgalactosamine residues and about 3 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0048] In certain embodiments, the isolated polysaccharide comprises about 96 N-acetylgalactosamine residues and about 4 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0049] In certain embodiments, the isolated polysaccharide comprises about 95 N-acetylgalactosamine residues and about 5 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0050] In certain embodiments, the isolated polysaccharide comprises about 94 N-acetylgalactosamine residues and about 6 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0051] In certain embodiments, the isolated polysaccharide comprises about 93 N-acetylgalactosamine residues and about 7 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0052] In certain embodiments, the isolated polysaccharide comprises about 92 N-acetylgalactosamine residues and about 8 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0053] In certain embodiments, the isolated polysaccharide comprises about 91 N-acetylgalactosamine residues and about 9 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0054] In certain embodiments, the isolated polysaccharide comprises about 90 N-acetylgalactosamine residues and about 10 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0055] In certain embodiments, the isolated polysaccharide comprises about 85 N-acetylgalactosamine residues and about 15 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0056] In certain embodiments, the isolated polysaccharide comprises about 80 N-acetylgalactosamine residues and about 20 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0057] In certain embodiments, the isolated polysaccharide comprises about 75 N-acetylgalactosamine residues and about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0058] In certain embodiments, the isolated polysaccharide comprises about 70 N-acetylgalactosamine residues and about 30 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0059] In certain embodiments, the isolated polysaccharide comprises about 65 N-acetylgalactosamine residues and about 35 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0060] In certain embodiments, the isolated polysaccharide comprises about 60 N-acetylgalactosamine residues and about 40 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0061] In certain embodiments, the isolated polysaccharide comprises about 55 N-acetylgalactosamine residues and about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0062] In certain embodiments, the isolated polysaccharide comprises about 50 N-acetylgalactosamine residues and about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0063] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 50 N-acetylgalactosamine residues and from about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0064] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 55 N-acetylgalactosamine residues and from about 0.1 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0065] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 75 N-acetylgalactosamine residues and from about 0.1 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0066] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99 to about 75 N-acetylgalactosamine residues and from about 1 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0067] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising about 95 to about 50 N-acetylgalactosamine residues and about 5 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0068] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising about 95 to about 55 N-acetylgalactosamine residues and about 5 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0069] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising about 95 to about 75 N-acetylgalactosamine residues and about 5 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0070] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising about 90 to about 50 N-acetylgalactosamine residues and about 10 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0071] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 90 to about 55 N-acetylgalactosamine residues and from about 10 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0072] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising about 90 to about 75 N-acetylgalactosamine residues and about 10 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0073] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 99.5 N-acetylgalactosamine residues and from about 0.1 to about 0.5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0074] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 99 N-acetylgalactosamine residues and from about 0.1 to about 1 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0075] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 98 N-acetylgalactosamine residues and from about 0.1 to about 2 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0076] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 97 N-acetylgalactosamine residues and from about 0.1 to about 3 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0077] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.9 to about 95 N-acetylgalactosamine residues and from about 0.1 to about 5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0078] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.8 to about 99.5 N-acetylgalactosamine residues and from about 0.2 to about 0.5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0079] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.8 to about 99 N-acetylgalactosamine residues and from about 0.2 to about 1 4-keto-N-acetyl-quinovosamine residue per 100 saccharide repeat units of the polysaccharide.

[0080] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.8 to about 98 N-acetylgalactosamine residues and from about 0.2 to about 2 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0081] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.8 to about 97 N-acetylgalactosamine residues and from about 0.2 to about 3 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0082] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.8 to about 95 N-acetylgalactosamine residues and from about 0.2 to about 5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0083] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.5 to about 99 N-acetylgalactosamine residues and from about 0.5 to about 1 4-keto-N-acetyl-quinovosamine residue per 100 saccharide repeat units of the polysaccharide.

[0084] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.5 to about 98 N-acetylgalactosamine residues and from about 0.5 to about 2 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0085] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.5 to about 97 N-acetylgalactosamine residues and from about 0.5 to about 3 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0086] In one embodiment, the present invention provides an isolated S. pneumoniae serotype 12F capsular polysaccharide comprising from about 99.5 to about 95 N-acetylgalactosamine residues and from about 0.5 to about 5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeat units of the polysaccharide.

[0087] In certain embodiments, the isolated polysaccharide has between 10 and 5,000 repeating units. In particular aspects, the isolated polysaccharide has between 50 and 4,500 repeating units. In particular aspects, the isolated polysaccharide has between 100 and 4,500 repeating units. In particular aspects, the isolated polysaccharide has between 150 and 2,000 repeating units.

[0088] Isolated capsular saccharides from S. pneumoniae serotype 12F are prepared by standard techniques known to those skilled in the art. Typically, capsular polysaccharides are produced by growing S. pneumoniae serotype 12F strains in culture media (e.g., soy-based media), and the polysaccharides are then prepared from the bacterial culture. Serotype 12F Streptococcus pneumoniae strains can be obtained from established culture collections (such as the Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA)) or from clinical specimens.

[0089] Populations of the organism (S. pneumoniae serotype 12F) are often expanded from seed vials to seed bottles and passaged through one or more seed fermenters of increasing volume until a production-scale fermentation volume is reached. At the end of the growth cycle, the cells are lysed and the lysate broth is then harvested for downstream (purification) processing (see, e.g., WO2006 / 110381 and WO2008 / 118752, U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2008 / 0102498, and US2008 / 0286838). Polysaccharides are typically purified through centrifugation, precipitation, ultrafiltration, and / or column chromatography (see, e.g., WO2006 / 110352, WO2008 / 118752, and WO2020 / 170190).

[0090] The isolated polysaccharides can be characterized by various parameters including, for example, the weight average molecular weight (Mw).

[0091] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) in combination with multi-angle laser light scattering detection (MALLS).

[0092] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 1000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 500 kDa. In one embodiment, the isolated capsular polysaccharide has a weight average molecular weight of 5 kDa to 400 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 300 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 200 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 5 kDa to 100 kDa.

[0093] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 1000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 500 kDa. In one embodiment, the isolated capsular polysaccharide has a weight average molecular weight of 50 kDa to 400 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 300 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 200 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 50 kDa to 100 kDa.

[0094] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 1000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 500 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 400 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 300 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 200 kDa.

[0095] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 1000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 500 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 400 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 200 kDa to 300 kDa.

[0096] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 300 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 300 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 300 kDa to 1000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 300 kDa to 500 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 300 kDa to 400 kDa.

[0097] In one embodiment, the isolated polysaccharide has a weight average molecular weight of 500 kDa to 5000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 500 kDa to 2000 kDa. In one embodiment, the isolated polysaccharide has a weight average molecular weight of 500 kDa to 1000 kDa. In a preferred embodiment, the isolated polysaccharide has a weight average molecular weight of 100 kDa to 500 kDa.

[0098] 2. Streptococcus pneumoniae serotype 12F glycoconjugates of the present invention As further described herein, the isolated polysaccharides described above can be activated (e.g., chemically activated) to make them reactive (e.g., with a linker or directly with a carrier protein) and then incorporated into glycoconjugates.

[0099] For purposes of the present invention, the term "glycoconjugate" refers to a sugar covalently attached to a carrier protein. In one embodiment, the sugar is directly linked to the carrier protein. In a second embodiment, the sugar is linked to the carrier protein through a spacer / linker.

[0100] In general, covalent conjugation of saccharides to carriers enhances the immunogenicity of saccharides by converting them from T-independent antigens to T-dependent antigens, thus enabling priming for immunological memory. Conjugation is particularly useful for pediatric vaccines.

[0101] Prior to activation, the size of the isolated polysaccharide can be reduced while preserving important features of the polysaccharide's structure. Mechanical or chemical sizing can be used. In one embodiment, the size of the isolated polysaccharide is reduced by chemical hydrolysis. The size of the isolated polysaccharide can also be reduced by mechanical homogenization. In one embodiment, the size of the isolated polysaccharide is reduced by high-pressure homogenization. In high-pressure homogenization, high shear rates are achieved by pumping the process stream through channels with sufficiently small dimensions. The shear rate can be increased by using higher homogenization pressures, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0102] In one embodiment, the present invention relates to a serotype 12F glycoconjugate prepared by a process comprising the steps of: a) reacting the isolated polysaccharide of Section 1 above with an activating agent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein.

[0103] In one embodiment, the serotype 12F glycoconjugates of the invention comprise a serotype 12F polysaccharide, wherein said polysaccharide has a weight average molecular weight (Mw) of 50 kDa to 1,000 kDa prior to conjugation.

[0104] In one embodiment, the serotype 12F glycoconjugates of the invention comprise a serotype 12F polysaccharide, wherein said polysaccharide prior to conjugation has a weight average molecular weight (Mw) of between 100 kDa and 600 kDa.

[0105] In one embodiment, the serotype 12F glycoconjugates of the invention comprise a serotype 12F polysaccharide, wherein said polysaccharide prior to conjugation has a weight average molecular weight (Mw) of between 150 kDa and 400 kDa.

[0106] In one embodiment, the serotype 12F glycoconjugates of the invention comprise a serotype 12F polysaccharide, wherein said polysaccharide prior to conjugation has a weight average molecular weight (Mw) of between 150 kDa and 300 kDa.

[0107] In some embodiments, the serotype 12F glycoconjugates of the invention have a weight average molecular weight (Mw) of between 250 kDa and 15,000 kDa.

[0108] In another embodiment, the serotype 12F glycoconjugate has a weight average molecular weight (Mw) of 500 kDa to 2,500 kDa.

[0109] In yet other embodiments, the serotype 12F glycoconjugates have a weight average molecular weight (Mw) of between 750 kDa and 2,500 kDa.

[0110] In a preferred embodiment, the serotype 12F glycoconjugates have a weight average molecular weight (Mw) of between 1,000 kDa and 2,500 kDa.

[0111] Another method for characterizing the serotype 12F glycoconjugates of the invention is to characterize the carrier protein (e.g., CRMP) to which the saccharide is conjugated. 197The degree of conjugation of a carrier protein depends on the number of lysine residues in the carrier protein, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent attachment to a polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of serotype 12F glycoconjugates of the invention is 2-15. In one embodiment, the degree of conjugation of serotype 12F glycoconjugates of the invention is 2-10. In one embodiment, the degree of conjugation of serotype 12F glycoconjugates of the invention is 3-5. In one embodiment, the degree of conjugation of serotype 12F glycoconjugates of the invention is 2-6. In one embodiment, the degree of conjugation of serotype 12F glycoconjugates of the invention is 4-10.

[0112] The serotype 12F glycoconjugates of the present invention may also be characterized by the sugar-to-carrier protein ratio (w / w). In some embodiments, the serotype 12F polysaccharide-to-carrier protein ratio (w / w) in the glycoconjugate is 0.5-3.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-2.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.8-1.2. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-1.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0-1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0-2.0. In a further embodiment, the sugar-to-carrier protein ratio (w / w) is 0.8-1.2. In preferred embodiments, the ratio of serotype 12F capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is a CRM 197 is.

[0113] The process for preparing serotype 12F glycoconjugates of the present invention may include the use of a reducing agent. Specifically, unreacted aldehyde groups after oxidation (specifically when using reductive amination, see below) may be capped using a suitable capping agent (reducing agent). In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0114] As shown in Example 2, the 4KQ (4-keto-N-acetyl-quinovosamine) residue is susceptible to reduction using NaBH4. Treatment of serotype 12F polysaccharide with NaBH4 specifically reduces position 4 of the 4KQ residue from a ketone / hydrate to an alcohol, converting residue 4KQ to a mixture of D-FucNAc and D-QuiNAc, characterized by the hydroxyl at position 4 in the axial and equatorial orientations, respectively, as illustrated in Figure 6.

[0115] Thus, in one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0116] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0117] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0118] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0119] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0120] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0121] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0122] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0123] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0124] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0125] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0126] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0127] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0128] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0129] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0130] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0131] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0132] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0133] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0134] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0135] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0136] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0137] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0138] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0139] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0140] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0141] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 4 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0142] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0143] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0144] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0145] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0146] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0147] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0148] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 10 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0149] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0150] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 10 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0151] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0152] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0153] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0154] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0155] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0156] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0157] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0158] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0159] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0160] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0161] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0162] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0163] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0164] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeat units of the polysaccharide.

[0165] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0166] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0167] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0168] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0169] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0170] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0171] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0172] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0173] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0174] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0175] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0176] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0177] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 4 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0178] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0179] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0180] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0181] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0182] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0183] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0184] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 10 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0185] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0186] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 10 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeat units of the polysaccharide.

[0187] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0188] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0189] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0190] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0191] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0192] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0193] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0194] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0195] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0196] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0197] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0198] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0199] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0200] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0201] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0202] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0203] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 2 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0204] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 2 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0205] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 2 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0206] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 2 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 2 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0207] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 3 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0208] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 3 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0209] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 3 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0210] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 3 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 3 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0211] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 4 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0212] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 4 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0213] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 4 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0214] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 4 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 4 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0215] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0216] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0217] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0218] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0219] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 7.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 7.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0220] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 7.5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 7.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0221] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 7.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0222] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 7.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 7.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0223] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 10 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0224] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 10 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 10 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0225] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 10 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 10 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0226] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.05 to about 0.1 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 0.1 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0227] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.05 to about 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0228] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.05 to about 1 N-acetyl-D-fucosamine (D-FucNAc) residue and from about 0.05 to about 1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0229] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.1 to about 0.2 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 0.2 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0230] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.1 to about 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0231] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising from about 0.1 to about 1 N-acetyl-D-fucosamine (D-FucNAc) residue and from about 0.1 to about 1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0232] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.05 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0233] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.1 N-acetyl-D-fucosamine (D-FucNAc) residue and about 0.1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0234] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0235] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 1 N-acetyl-D-fucosamine (D-FucNAc) residue and about 1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0236] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about two N-acetyl-D-fucosamine (D-FucNAc) residues and about two N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0237] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 3 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0238] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0239] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 7 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0240] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 12 N-acetyl-D-fucosamine (D-FucNAc) residues and about 12 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0241] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0242] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 13 N-acetyl-D-fucosamine (D-FucNAc) residues and about 13 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0243] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 14 N-acetyl-D-fucosamine (D-FucNAc) residues and about 14 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0244] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0245] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0246] In one embodiment, a serotype 12F glycoconjugate of the invention comprises a serotype 12F capsular polysaccharide comprising about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0247] The serotype 12F glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (i.e., non-covalently bound, adsorbed, or entrapped in or with) the glycoconjugate.

[0248] In a preferred embodiment, the serotype 12F glycoconjugates comprise less than about 50% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide. In a preferred embodiment, the serotype 12F glycoconjugates comprise less than about 25% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide. In a further preferred embodiment, the serotype 12F glycoconjugates comprise less than about 20% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide. In a further preferred embodiment, the serotype 12F glycoconjugates comprise less than about 15% free serotype 12F polysaccharide relative to the total amount of serotype 12F polysaccharide.

[0249] Serotype 12F glycoconjugates also exhibit a molecular size distribution (K d ) can also be characterized. Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Large molecules that are excluded from the pores in the media elute more quickly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by sugar assay. K d To determine the fraction, the column is calibrated to determine the fraction at which the molecule is completely excluded (V0), (K d =0), and maximum hold (V i ), (K dEstablish the fraction that represents the fraction at which the specified sample characteristic is reached (V e ) is the formula, K d =(V e -V0) / (V i -V0) by K d Related to.

[0250] In a preferred embodiment, at least 30% of serotype 12F glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 40% of the glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 60% of serotype 12F glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 50% to 80% of serotype 12F glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 65% to 80% of serotype 12F glycoconjugates have a K of 0.3 or less in a CL-4B column. d It has.

[0251] Carrier proteins Another component of the glycoconjugates of the present disclosure is a carrier protein to which the saccharide is conjugated. The term "protein carrier" or "carrier protein" or "carrier" refers to any protein molecule that can be conjugated to an antigen (such as a capsular polysaccharide) against which an immune response is desired.

[0252] Conjugation to a carrier can enhance the immunogenicity of an antigen. Protein carriers for antigens can be toxins, toxoids, or any mutant cross-reactive material (CRM) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus. In one embodiment, the carrier protein is a CRM. 197CRM from C. diphtheriae strain C7(β197) producing protein 197 This strain has ATCC accession number 53281. 197 Methods for producing β-glucan are described in U.S. Patent No. 5,614,382. Alternatively, protein carriers or fragments or epitopes of other immunogenic proteins can be used. For example, hapten antigens can be coupled to T cell epitopes of bacterial toxins, toxoids, or CRMs. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in International Patent Application WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), or Haemophilus influenzae protein D, can also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.

[0253] In a preferred embodiment, the carrier protein of the serotype 12F glycoconjugates of the invention is TT (tetanus toxoid), DT (diphtheria toxoid), a DT mutant (such as CRM197), or C5a peptidase from Streptococcus (SCP).

[0254] In a preferred embodiment, the carrier protein of the serotype 12F glycoconjugate of the present invention is selected from the group consisting of TT (tetanus toxoid), DT (diphtheria toxoid), DT mutants (such as CRM197), and C5a peptidase from Streptococcus (SCP).

[0255] In one embodiment, the carrier protein of the serotype 12F capsular polysaccharide glycoconjugate is DT (diphtheria toxoid). In another embodiment, the carrier protein of the serotype 12F capsular polysaccharide glycoconjugate is TT (tetanus toxoid).

[0256] In another embodiment, the carrier protein of the serotype 12F capsular polysaccharide glycoconjugate is PD (H. influenzae protein D, see for example EP0594610B).

[0257] In a preferred embodiment, the carrier protein of the serotype 12F capsular polysaccharide glycoconjugate is CRM 197 is.

[0258] As described herein above, the number of lysine residues in a carrier protein that are conjugated to a saccharide can be characterized as a range of conjugated lysines. For example, in a given immunogenic composition, the number of lysine residues in a carrier protein that are conjugated to a saccharide can be characterized as a range of conjugated lysines. 197 may contain lysine residues covalently linked to 1 to 15 of the 39 sugars. Another way to express this parameter is the CRM 197 About 2.5% to about 40% of the lysines are covalently linked to sugars. For example, in a given immunogenic composition, CRM 197 may contain 1 to 20 of the 39 lysine residues covalently linked to 12F sugars. Another way to express this parameter is the CRM 197 Approximately 2.5% to 50% of the lysines are covalently bound to 12F sugars.

[0259] 3. Methods for Producing the Streptococcus pneumoniae Serotype 12F Glycoconjugates of the Present Invention In one embodiment, serotype 12F glycoconjugates of the invention are prepared using reductive amination.

[0260] Reductive amination involves two steps: (1) oxidizing (activating) purified sugars and (2) oxidizing activated sugars and carrier proteins (e.g., CRMs). 197, TT, or SCP) to form glycoconjugates (see, e.g., WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, WO2018 / 156491).

[0261] As mentioned above, oxidation can be preceded by sizing of the polysaccharide to a target molecular weight (MW) range.

[0262] Thus, in one embodiment, the isolated 12F polysaccharide is sized prior to oxidation, hi one embodiment, the isolated 12F polysaccharide is sized to any of the target molecular weight (MW) ranges defined above.

[0263] In one embodiment, the serotype 12F glycoconjugates of the invention are prepared by a process comprising the steps of: a) reacting a serotype 12F saccharide with a stable nitroxyl radical compound and an oxidizing agent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein.

[0264] In one embodiment, the stable nitroxyl radical compound is a molecule bearing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without affecting secondary hydroxyl groups. More preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without overoxidation to carboxyl groups. In one embodiment, the stable nitroxyl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, or 4-amino-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO. In one embodiment, the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO.In a further aspect, the stable nitroxyl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL. In a further embodiment, the stable nitroxyl radical compound is selected from the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, and 3-cyano-PROXYL. In one embodiment, the oxidizing agent is a molecule bearing an N-halo moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols in the presence of a nitroxyl radical compound. In one aspect, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. In one embodiment, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0265] In one embodiment, the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0266] In one embodiment, step a) of the reaction is carried out in an aqueous solvent. In another embodiment, step a) is carried out in an aprotic solvent. In one embodiment, step a) is carried out in DMSO (dimethyl sulfoxide), dimethylacetamide (DMA), sulfolane, N-methyl-2-pyrrolidone (NMP), hexamethylphosphoramide (HMPA), or DMF (dimethylformamide) solvent. In one embodiment, step a) is carried out in DMSO (dimethyl sulfoxide).

[0267] In one embodiment, the sugar is reacted with 0.1 to 10 molar equivalents of oxidizing agent. Preferably, the sugar is reacted with 0.2 to 5, 0.5 to 2.5, or 0.5 to 1.5 molar equivalents of oxidizing agent. In one embodiment, the polysaccharide is reacted with about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, or 5 molar equivalents of oxidizing agent.

[0268] In one embodiment, the stable nitroxyl radical compound is present in a catalytic amount. In one embodiment, the sugar is reacted with less than about 0.3 molar equivalents of the stable nitroxyl radical compound. In one embodiment, the sugar is reacted with less than about 0.005 molar equivalents of the stable nitroxyl radical compound. In one embodiment, the sugar is reacted with about 0.005, 0.01, 0.05, or 0.1 molar equivalents of the stable nitroxyl radical compound.

[0269] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent, which in one embodiment is sodium borohydride (NaBH4).

[0270] In one embodiment, capping is achieved by combining the product of step c) with 0.5 to 20 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 1 to 15 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 0.5 to 5 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 0.75 to 3 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 1 molar equivalent of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 2 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 3 molar equivalents of sodium borohydride.

[0271] In one embodiment, the serotype 12F glycoconjugates of the present invention comprise: (a) reacting the isolated serotype 12 F polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate; It is prepared by a process comprising:

[0272] In one embodiment, the serotype 12F glycoconjugates of the present invention comprise: (a) reacting the isolated serotype 12 F polysaccharide with an oxidizing agent; (a') quenching the oxidation reaction by adding a quenching agent; (b) combining the activated polysaccharide of step (a') with a carrier protein; (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a glycoconjugate; It is prepared by a process comprising:

[0273] After the oxidation step (a), the saccharide is said to be activated and is called an "activated polysaccharide."

[0274] In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. For purposes of the present invention, the term "periodate" includes both periodate and periodic acid. This term also refers to metaperiodate (IO4 - ) and orthoperiodate (IO6 5- ), as well as various salts of periodate (e.g., sodium periodate and potassium periodate).

[0275] In one embodiment, the oxidizing agent is orthoperiodate.

[0276] In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the periodate used for oxidation is metaperiodate. In one embodiment, the periodate used for oxidation is sodium metaperiodate.

[0277] When a polysaccharide reacts with periodate, periodate oxidizes vicinal hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of C-C bonds. Thus, the term "reacting a polysaccharide with periodate" includes the oxidation of vicinal hydroxyl groups by periodate.

[0278] In one embodiment, step a) comprises reacting the polysaccharide with 0.01 to 2 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.05 to 1.5 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.1 to 1.0 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.01 to 0.5 molar equivalents of periodate. In one embodiment, step a) comprises reacting the polysaccharide with 0.1 to 0.5 molar equivalents of periodate.

[0279] In one embodiment, the quenching agent in step a') is selected from a vicinal diol, a 1,2-amino alcohol, an amino acid, glutathione, a sulfite, a bisulfate, a dithionite, a metabisulfite, a thiosulfate, a phosphite, a hypophosphite, or a phosphorous acid.

[0280] In one embodiment, the quenching agent is a 1,2-amino alcohol of formula (I):

[0281] [ka]

[0282] [In the formula, R 1 is selected from H, methyl, ethyl, propyl, or isopropyl.

[0283] In one embodiment, the quenching agent is selected from sodium and potassium salts of sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphite, hypophosphite, or phosphorous acid.

[0284] In one embodiment, the quenching agent is an amino acid, which may be selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0285] In one embodiment, the quenching agent is a sulfite, such as bisulfate, dithionite, metabisulfite, or thiosulfate.

[0286] In one embodiment, the quenching agent is a compound containing two vicinal hydroxyl groups (vicinal diol), ie, two hydroxyl groups covalently bonded to two adjacent carbon atoms.

[0287] Preferably, the quenching agent is a compound of formula (II):

[0288] [ka]

[0289] [In the formula, R 1 and R 2 are each independently selected from H, methyl, ethyl, propyl, or isopropyl.

[0290] In a preferred embodiment, the quenching agent is glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol, or butane-2,3-diol, or ascorbic acid. In a more preferred embodiment, the quenching agent is butane-2,3-diol.

[0291] In a preferred embodiment, the oxidation degree of the active serotype 12F polysaccharide is 2-30.

[0292] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. In one embodiment, the reduction reaction (c) is carried out in an aprotic solvent.

[0293] In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0294] In one embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). In one embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl formamide (DMF). In one embodiment, the reduction reaction (c) is carried out in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0295] In one embodiment, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethyl formamide) solvent. In one embodiment, the reduction reaction (c) is carried out in DMSO (dimethyl sulfoxide) solvent.

[0296] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, an amine borane such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i The reducing agent is PrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB). In one embodiment, the reducing agent is sodium triacetoxyborohydride. In a preferred embodiment, the reducing agent is sodium cyanoborohydride. In one embodiment, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439).

[0297] In one embodiment, 1.0 to 20 molar equivalents of reducing agent are used in step c). In one embodiment, 2 to 15 molar equivalents of reducing agent are used in step c). In one embodiment, 5 to 15 molar equivalents of reducing agent are used in step c).

[0298] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent, which in one embodiment is sodium borohydride (NaBH4).

[0299] In one embodiment, capping is achieved by combining the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 2 to 15 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by combining the product of step c) with 5 to 10 molar equivalents of sodium borohydride.

[0300] Following conjugation with a carrier protein, the glycoconjugates can be purified (enriched in terms of the amount of glyco-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one embodiment, the process of producing the glycoconjugates of the present invention includes a step of purifying the glycoconjugates after they have been produced.

[0301] 4. Immunogenic composition In one embodiment, the invention relates to an immunogenic composition comprising the S. pneumoniae serotype 12F saccharide of the invention.

[0302] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention.

[0303] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention, wherein the composition comprises from 1 to 25 different glycoconjugates.

[0304] In one embodiment, the invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 12F glycoconjugates of the invention, comprising 1 to 25 glycoconjugates from different serotypes of S. pneumoniae (1 to 25 pneumococcal conjugates). In one embodiment, the invention relates to an immunogenic composition comprising glycoconjugates from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises glycoconjugates from 16 or 20 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition is a 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 14-, 15-, 16-, 17-, 18-, or 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0305] In one embodiment, the invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 12F glycoconjugates of the invention, comprising 26 to 45 glycoconjugates from different serotypes of S. pneumoniae (26 to 45 pneumococcal conjugates). In one embodiment, the invention relates to an immunogenic composition comprising glycoconjugates from 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises glycoconjugates from 35 or 45 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition is a 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-, or 45-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 40-, 41-, 42-, 43-, 44-, or 45-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 40-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 41-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 42-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 43-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 44-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 45-valent pneumococcal conjugate composition.

[0306] In one embodiment, the present invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 12F glycoconjugates of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

[0307] In one embodiment, the immunogenic composition additionally comprises glycoconjugates from S. pneumoniae serotypes 1, 5, and 7F.

[0308] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 3.

[0309] In one embodiment, any of the above immunogenic compositions additionally comprises glycoconjugates from S. pneumoniae serotypes 6A and 19A.

[0310] In one embodiment, any of the above immunogenic compositions additionally comprises glycoconjugates from S. pneumoniae serotypes 22F and 33F.

[0311] In one embodiment, any of the above immunogenic compositions additionally comprises glycoconjugates from S. pneumoniae serotypes 8, 10A, 11A, and 15B.

[0312] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 2.

[0313] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 9N.

[0314] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 17F.

[0315] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 20.

[0316] In one embodiment, any of the above immunogenic compositions additionally comprises a glycoconjugate from S. pneumoniae serotype 15C.

[0317] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. In one embodiment, the immunogenic composition is an octavalent pneumococcal conjugate composition.

[0318] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. In one embodiment, the immunogenic composition is an 11-valent pneumococcal conjugate composition.

[0319] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 16-valent pneumococcal conjugate composition.

[0320] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0321] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 12F glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15C, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0322] In a preferred embodiment, the saccharides are each individually conjugated to a different molecule of the protein carrier (each molecule of the protein carrier has only one type of saccharide conjugated to it). In said embodiment, the capsular saccharides are said to be individually conjugated to the carrier protein. Preferably, all the glycoconjugates of the immunogenic composition are individually conjugated to the carrier protein.

[0323] In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 12F is a CRM 197 It is conjugated with

[0324] In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 22F is a CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 33F is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 15B is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 10A is conjugated to CRM 197In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 11A is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 8 is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 1, 5, and 7F are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 6A and 19A are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 3 is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 15C is conjugated to CRM 197 It is conjugated with

[0325] In one embodiment of any of the above immunogenic compositions, the glycoconjugates of any of the above immunogenic compositions all individually contain a CRM 197 It is conjugated with

[0326] In one embodiment, the immunogenic composition comprises 8 to 20 different serotypes of S. pneumoniae, hi one embodiment, the immunogenic composition comprises 21 to 45 different serotypes of S. pneumoniae.

[0327] The compositions of the present invention may contain small amounts of free carrier. When a given carrier protein is present in both free and conjugated forms in the compositions of the present invention, the unconjugated form preferably represents no more than 5% of the total amount of carrier protein in the entire composition, more preferably less than 2% by weight.

[0328] The immunogenic compositions of the present disclosure can be used to protect or treat humans susceptible to bacterial infections, such as those caused by Streptococcus pneumoniae bacteria, by administering the immunogenic compositions via systemic, dermal, or mucosal routes, or to generate polyclonal or monoclonal antibody preparations that can be used to confer passive immunity to another subject. These administrations can include injection via intramuscular, intraperitoneal, intradermal, or subcutaneous routes, or via oral / dietary, respiratory, or genitourinary tract mucosal administration. The immunogenic compositions can also be used to generate antibodies that are functional as measured by bacterial killing in either animal efficacy models or via opsonophagocytic killing assays.

[0329] Optimal amounts of components for a particular immunogenic composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects can receive one or several booster immunizations adequately spaced apart.

[0330] In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise two adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An antigen may act primarily as a delivery system, act primarily as an immunomodulator, or have both powerful features. Suitable adjuvants include those suitable for use in mammals, including humans.

[0331] Examples of known suitable delivery system-type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0332] In one embodiment, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate as an adjuvant.

[0333] Additional exemplary adjuvants for enhancing the effectiveness of the immunogenic compositions disclosed herein include, but are not limited to, (1) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components), such as (a) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic®-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to create a larger particle size emulsion, and (b) 2% squalene, 0.2% Tween 80, and monophosphorylipid A. A) the RIBI™ adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), which contains one or more bacterial cell wall components such as (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), ABISCO® (Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum) (2) saponin adjuvants such as saponin adjuvants (e.g., saponin adjuvants such as saponin adjuvants, ...(6) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions (see, for example, EP 0835318, EP 0735898, EP 0761231); (7) polyoxyethylene ethers or polyoxyethylene esters (see, for example, WO 99 / 52549); (8) combinations of polyoxyethylene sorbitan ester surfactants with octoxynol (see, for example, WO 01 / 21207) or polyoxyethylene alkyl ether or ester surfactants with octoxynol, etc. (9) saponin and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO00 / 62800); (10) immunostimulants and metal salt particles (see, e.g., WO00 / 23105); (11) saponin and oil-in-water emulsions (e.g., WO99 / 11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol) (e.g., WO98 / 57659); and (13) other substances that act as immunostimulants to enhance the effectiveness of the composition. Examples of muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0334] In one embodiment of the present invention, the immunogenic composition disclosed herein comprises a CpG oligonucleotide as an adjuvant. As used herein, CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN); therefore, unless otherwise specified, these terms are used interchangeably. An immunostimulatory CpG oligodeoxynucleotide contains one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally within a specific preferred base context. The methylation state of a CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide that contains a 5' unmethylated cytosine linked to a 3' guanine by a phosphate bond and activates the immune system through binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotides may contain one or more methylated CpG dinucleotides, which activate the immune system through TLR9, but not as potently as when the CpG motifs are unmethylated. The CpG immunostimulatory oligonucleotides may contain one or more palindromic structures, which in turn may encompass CpG dinucleotides. CpG oligonucleotides are described in several issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068.

[0335] 5. Uses of the Immunogenic Compositions of the Invention The S. pneumoniae serotype 12F saccharides or S. pneumoniae serotype 12F glycoconjugates disclosed herein may be used as antigens, for example they may be part of a vaccine.

[0336] Thus, in one embodiment, the immunogenic composition of the invention is for use as a pharmaceutical.

[0337] In one embodiment, the immunogenic composition of the invention is for use as a vaccine.

[0338] Thus, in one embodiment, the immunogenic compositions described herein are for use in generating an immune response in a subject. In one aspect, the subject is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog. In one aspect, the subject is a human.

[0339] The immunogenic compositions described herein may be used in therapeutic or prophylactic methods to prevent, treat, or ameliorate a bacterial infection, disease, or condition in a subject. Specifically, the immunogenic compositions described herein may be used to prevent, treat, or ameliorate a S. pneumoniae serotype 12F infection, disease, or condition in a subject.

[0340] Accordingly, in one aspect, the present disclosure provides a method of preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotype 12F in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present disclosure.

[0341] In some such embodiments, the infection, disease, or condition is pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, or brain abscess.

[0342] In some such embodiments, the infection, disease, or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.

[0343] In one embodiment, the present disclosure provides a method of inducing an immune response against S. pneumoniae serotype 12F in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention. In one aspect, the subject is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog. In one aspect, the subject is a human.

[0344] In one embodiment, the immunogenic compositions disclosed herein are for use as vaccines. In such embodiments, the immunogenic compositions described herein may be used to prevent S. pneumoniae serotype 12F infection in a subject. Accordingly, in one aspect, the present invention provides a method for preventing S. pneumoniae serotype 12F infection in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present disclosure. In some such embodiments, the infection is pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, or brain abscess. In one aspect, the subject is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog. In one aspect, the subject is a human. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In one aspect, the subject is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog. In one aspect, the subject is a human.

[0345] The immunogenic compositions of the present disclosure can be used to protect or treat humans susceptible to S. pneumoniae serotype 12F infection by administering the immunogenic compositions via systemic or mucosal routes. In one embodiment, the immunogenic compositions of the present invention are administered via intramuscular, intraperitoneal, intradermal, or subcutaneous routes. In one embodiment, the immunogenic compositions of the present invention are administered via intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In one embodiment, the immunogenic compositions of the present invention are administered via intramuscular or subcutaneous injection. In one embodiment, the immunogenic compositions of the present invention are administered via intramuscular injection. In one embodiment, the immunogenic compositions of the present invention are administered via subcutaneous injection.

[0346] 6.Analysis method In one embodiment, the present invention relates to a method for detecting the presence of 4-keto-N-acetyl-quinovosamine residues in isolated S. pneumoniae serotype 12 F polysaccharide, comprising the steps of a) isolating S. pneumoniae serotype 12 F polysaccharide, and b) detecting the presence of 4-keto-N-acetyl-quinovosamine residues in said polysaccharide.

[0347] In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by NMR or mass spectrometry (MS). In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by NMR. In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by 1D NMR. In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by 1D NMR. 1 H or 1D 13The presence of the 4-keto-N-acetyl-quinovosamine residue is detected by C NMR. In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by 2D NMR. In one embodiment, the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), nuclear Overhauser effect spectroscopy (NOESY), correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0348] In one embodiment, the presence of a 4-keto-N-acetyl-quinovosamine residue is 1 H, 2D 1 H- 13 C heteronuclide single quantum coherence spectroscopy (HSQC), 2D 1 H- 13 C Heteronuclide Multiple Quantum Correlation Spectroscopy (HMBC), 2D 1 H- 13 C Nuclear Overhauser Effect Spectroscopy (NOESY), 2D 1 H- 13 C correlation spectroscopy (COSY), 2D 1 H- 13 C total correlation spectroscopy (TOCSY), 2D 1 H- 13 C heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY), or 1D 13 Detected by C NMR.

[0349] In a preferred embodiment, the presence of a 4-keto-N-acetyl-quinovosamine residue is 1 H, 2D 1 H- 13 C heteronuclear single quantum coherence spectroscopy (HSQC), or 1D 13 Detected by C NMR.

[0350] In one embodiment, the presence of a 4-keto-N-acetyl-quinovosamine residue is 1 H- 13 Detected by C heteronuclear single quantum coherence spectroscopy (HSQC).

[0351] In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by mass spectrometry (MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0352] In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of 4-keto-N-acetyl-quinovosamine residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0353] In one embodiment, the present invention relates to a method for determining the amount of 4-keto-N-acetyl-quinovosamine residues in isolated S. pneumoniae serotype 12F polysaccharide, comprising the steps of a) isolating S. pneumoniae serotype 12F polysaccharide; and b) measuring the amount of 4-keto-N-acetyl-quinovosamine residues in said polysaccharide.

[0354] In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by NMR. In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by 1D NMR. In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by 1D NMR. 1 H or 1D 13 In a preferred embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by 1D NMR. 1 In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by 1D NMR. 1 It is determined by integration or deconvolution of the H spectrum.

[0355] In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by 2D NMR. 1 H- 13 C Determined by cross-peak integration of HSQC spectra.

[0356] In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by mass spectrometry (MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by tandem mass spectrometry (MS / MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0357] In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the amount of 4-keto-N-acetyl-quinovosamine residues is determined by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0358] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in reduced Streptococcus pneumoniae (S. pneumoniae) serotype 12 F polysaccharide, comprising the steps of a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent, and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in the reduced polysaccharide.

[0359] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by 2D NMR.

[0360] In a preferred embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues contributes to the 2D 1 H- 13 Detected by C HSQC NMR.

[0361] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0362] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0363] In one embodiment, the reducing agent is sodium borohydride (NaBH4).

[0364] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with an oxidizing agent. In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. In one embodiment, the oxidizing agent is orthoperiodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the oxidizing agent is metaperiodate. In a preferred embodiment, the oxidizing agent is sodium metaperiodate.

[0365] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with a stable nitroxyl radical compound and an oxidizing agent. In one aspect, the stable nitroxyl radical compound is a molecule bearing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without affecting secondary hydroxyl groups. More preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without overoxidation to carboxyl groups. In one embodiment, the stable nitroxyl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, or 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. In one embodiment, the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO.In a further aspect, the stable nitroxyl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL. In a further embodiment, the stable nitroxyl radical compound is selected from the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, and 3-cyano-PROXYL. In one embodiment, the oxidizing agent is a molecule bearing an N-halo moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols in the presence of a nitroxyl radical compound. In one aspect, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. In one embodiment, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0366] In one embodiment, the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0367] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in reduced Streptococcus pneumoniae (S. pneumoniae) serotype 12 F polysaccharide, comprising the steps of a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent, and b) detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in the reduced polysaccharide.

[0368] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by 2D NMR.

[0369] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), correlation spectroscopy (COSY), and / or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0370] In a preferred embodiment, the presence of an N-acetyl-D-quinovosamine (D-QuiNAc) residue is 1 H- 13 Detected by C HSQC NMR.

[0371] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0372] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0373] In one embodiment, the reducing agent is sodium borohydride (NaBH4).

[0374] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with an oxidizing agent. In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. In one embodiment, the oxidizing agent is orthoperiodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the oxidizing agent is metaperiodate. In a preferred embodiment, the oxidizing agent is sodium metaperiodate.

[0375] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with a stable nitroxyl radical compound and an oxidizing agent. In one aspect, the stable nitroxyl radical compound is a molecule bearing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without affecting secondary hydroxyl groups. More preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without overoxidation to carboxyl groups. In one embodiment, the stable nitroxyl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, or 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. In one embodiment, the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO.In a further aspect, the stable nitroxyl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL. In a further embodiment, the stable nitroxyl radical compound is selected from the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, and 3-cyano-PROXYL. In one embodiment, the oxidizing agent is a molecule bearing an N-halo moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols in the presence of a nitroxyl radical compound. In one aspect, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. In one embodiment, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0376] In one embodiment, the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0377] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in reduced Streptococcus pneumoniae (S. pneumoniae) serotype 12 F polysaccharide, comprising the steps of a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent, and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in the reduced polysaccharide.

[0378] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0379] In a preferred embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by 2D-NMR.

[0380] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by size exclusion chromatography coupled with mass spectrometry (SEC / MS).

[0381] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by liquid chromatography mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by capillary electrophoresis mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by hydrophilic interaction liquid chromatography mass spectrometry (HILIC-LC / MS).

[0382] In one embodiment, the reducing agent is sodium borohydride (NaBH4).

[0383] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with an oxidizing agent. In one embodiment, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes. In one embodiment, the oxidizing agent is periodate. In one embodiment, the oxidizing agent is orthoperiodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In one embodiment, the oxidizing agent is metaperiodate. In a preferred embodiment, the oxidizing agent is sodium metaperiodate.

[0384] In one embodiment, the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with a stable nitroxyl radical compound and an oxidizing agent. In one aspect, the stable nitroxyl radical compound is a molecule bearing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without affecting secondary hydroxyl groups. More preferably, the molecule has the ability to selectively oxidize primary alcohols to aldehyde groups in the presence of an oxidizing agent without overoxidation to carboxyl groups. In one embodiment, the stable nitroxyl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, or 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. In one embodiment, the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO.In a further aspect, the stable nitroxyl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL. In a further embodiment, the stable nitroxyl radical compound is selected from the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, 5-DOXYL-methyl stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, and 3-cyano-PROXYL. In one embodiment, the oxidizing agent is a molecule bearing an N-halo moiety. Preferably, the molecule has the ability to selectively oxidize primary alcohols in the presence of a nitroxyl radical compound. In one aspect, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. In one embodiment, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0385] In one embodiment, the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0386] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of a) preparing S. pneumoniae serotype 12F glycoconjugates, and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0387] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by 2D NMR.

[0388] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), correlation spectroscopy (COSY), and / or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0389] In a preferred embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues contributes to the 2D 1 H- 13 Detected by C HSQC NMR.

[0390] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by size exclusion chromatography coupled with mass spectrometry (SEC / MS).

[0391] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by liquid chromatography mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by capillary electrophoresis mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0392] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of a) preparing S. pneumoniae serotype 12F glycoconjugates, and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0393] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by 2D NMR.

[0394] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), correlation spectroscopy (COSY), and / or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0395] In a preferred embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues contributes to the 2D 1 H- 13 Detected by C HSQC NMR.

[0396] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by size exclusion chromatography coupled with mass spectrometry (SEC / MS).

[0397] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by liquid chromatography mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by capillary electrophoresis mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by hydrophilic interaction liquid chromatography mass spectrometry (HILIC-LC / MS).

[0398] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of a) preparing S. pneumoniae serotype 12F glycoconjugates, and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in said glycoconjugates.

[0399] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by 2D NMR.

[0400] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), correlation spectroscopy (COSY), and / or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0401] In a preferred embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues contributes to the 2D 1 H- 13 Detected by C HSQC NMR.

[0402] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by mass spectrometry (MS). The presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0403] In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0404] In one embodiment, the present invention relates to a method for detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of a) preparing S. pneumoniae serotype 12F glycoconjugates, and b) detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0405] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR. In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by 2D NMR.

[0406] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple quantum correlation spectroscopy (HMBC), correlation spectroscopy (COSY), and / or heteronuclear single quantum coherence spectroscopy-total correlation spectroscopy (HSQC-TOCSY).

[0407] In a preferred embodiment, the presence of an N-acetyl-D-quinovosamine (D-QuiNAc) residue is 1 H- 13 Detected by C HSQC NMR.

[0408] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by tandem mass spectrometry (MS / MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or ion mobility spectrometry-mass spectrometry (IMS / MS or IMMS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by a combination of size exclusion chromatography and mass spectrometry (SEC / MS).

[0409] In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by gas chromatography-mass spectrometry (GC-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by capillary electrophoresis-mass spectrometry (CE-MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by ion mobility spectrometry-mass spectrometry (IMS / MS). In one embodiment, the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC / MS).

[0410] 7. The present invention also provides the following embodiments, as defined in numbered paragraphs 1-296 below: 1. An isolated polysaccharide having the following repeating unit:

[0411] [ka] where n represents the number of repeating units and X represents either N-acetylgalactosamine or 4-keto-N-acetyl-quinovosamine.

[0412] 2. The isolated polysaccharide of paragraph 1, comprising from about 99.9 to about 50 N-acetylgalactosamine residues and from about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0413] 3. The isolated polysaccharide of paragraph 1, comprising from about 99.8 to about 50 N-acetylgalactosamine residues and from about 0.2 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0414] 4. The isolated polysaccharide of paragraph 1, comprising from about 99.1 to about 50 N-acetylgalactosamine residues and from about 0.9 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0415] 5. The isolated polysaccharide of paragraph 1, comprising about 99 to about 50 N-acetylgalactosamine residues and about 1 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0416] 6. The isolated polysaccharide of paragraph 1, comprising about 95 to about 50 N-acetylgalactosamine residues and about 5 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0417] 7. The isolated polysaccharide of paragraph 1, comprising about 90 to about 50 N-acetylgalactosamine residues and about 10 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0418] 8. The isolated polysaccharide of paragraph 1, comprising from about 99.9 to about 55 N-acetylgalactosamine residues and from about 0.1 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0419] 9. The isolated polysaccharide of paragraph 1, comprising from about 99.1 to about 55 N-acetylgalactosamine residues and from about 0.9 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0420] 10. The isolated polysaccharide of paragraph 1, comprising about 90 to about 55 N-acetylgalactosamine residues and about 10 to about 45 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0421] 11. The isolated polysaccharide of paragraph 1, comprising from about 99.9 to about 75 N-acetylgalactosamine residues and from about 0.1 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0422] 12. The isolated polysaccharide of paragraph 1, comprising about 99 to about 75 N-acetylgalactosamine residues and about 1 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0423] 13. The isolated polysaccharide of paragraph 1, comprising about 90 to about 75 N-acetylgalactosamine residues and about 10 to about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0424] 14. The isolated polysaccharide of paragraph 1, comprising about 99.9 to about 99.5 N-acetylgalactosamine residues and about 0.1 to about 0.5 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0425] 15. The isolated polysaccharide of paragraph 1, comprising about 99.9 to about 99 N-acetylgalactosamine residues and about 0.1 to about 1 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0426] 16. The isolated polysaccharide of paragraph 1, comprising about 99.9 to about 98 N-acetylgalactosamine residues and about 0.1 to about 2 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0427] 17. The isolated polysaccharide of paragraph 1, comprising about 99.8 to about 99.5 N-acetylgalactosamine residues and about 0.2 to about 0.5 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0428] 18. The isolated polysaccharide of paragraph 1, comprising about 99.8 to about 99 N-acetylgalactosamine residues and about 0.2 to about 1 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0429] 19. The isolated polysaccharide of paragraph 1, comprising about 99.9 N-acetylgalactosamine residues and about 0.1 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0430] 20. The isolated polysaccharide of paragraph 1, comprising about 99.8 N-acetylgalactosamine residues and about 0.2 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0431] 21. The isolated polysaccharide of paragraph 1, comprising about 80 N-acetylgalactosamine residues and about 20 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0432] 22. The isolated polysaccharide of paragraph 1, comprising about 75 N-acetylgalactosamine residues and about 25 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0433] 23. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 99.9 to about 50 N-acetylgalactosamine residues and about 0.1 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0434] 24. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising from about 99.9 to about 55 N-acetylgalactosamine residues and from about 0.1 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0435] 25. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising from about 99.9 to about 75 N-acetylgalactosamine residues and from about 0.1 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0436] 26. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 99 to about 75 N-acetylgalactosamine residues and about 1 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0437] 27. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide containing about 95 to about 50 N-acetylgalactosamine residues and about 5 to about 50 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0438] 28. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide containing about 95 to about 55 N-acetylgalactosamine residues and about 5 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0439] 29. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 95 to about 75 N-acetylgalactosamine residues and about 5 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0440] 30. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide containing about 90 to about 50 N-acetylgalactosamine residues and about 10 to about 50 4-keto-N-acetyl-quinovosamine residues for every 100 saccharide repeat units of the polysaccharide.

[0441] 31. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 90 to about 55 N-acetylgalactosamine residues and about 10 to about 45 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0442] 32. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide containing about 90 to about 75 N-acetylgalactosamine residues and about 10 to about 25 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0443] 33. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 99.9 to about 99.5 N-acetylgalactosamine residues and about 0.1 to about 0.5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0444] 34. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 99.9 to about 99 N-acetylgalactosamine residues and about 0.1 to about 1 4-keto-N-acetyl-quinovosamine residue per 100 saccharide repeating units of the polysaccharide.

[0445] 35. An isolated Streptococcus pneumoniae (S. pneumoniae) serotype 12F capsular polysaccharide comprising about 99.8 to about 99.5 N-acetylgalactosamine residues and about 0.2 to about 0.5 4-keto-N-acetyl-quinovosamine residues per 100 saccharide repeating units of the polysaccharide.

[0446] 36. The isolated polysaccharide of any one of paragraphs 1 to 35, having between 10 and 5,000 repeating units.

[0447] 37. The isolated polysaccharide of any one of paragraphs 1 to 35, having between 50 and 4,500 repeating units.

[0448] 38. The isolated polysaccharide of any one of paragraphs 1 to 35, having between 100 and 4,500 repeating units.

[0449] 39. The isolated polysaccharide of any one of paragraphs 1 to 35, having between 150 and 2,000 repeating units.

[0450] 40. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight average molecular weight of 5 kDa to 5000 kDa.

[0451] 41. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight-average molecular weight of 5 kDa to 2000 kDa.

[0452] 42. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight average molecular weight of 50 kDa to 1000 kDa.

[0453] 43. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight-average molecular weight of 50 kDa to 300 kDa.

[0454] 44. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight-average molecular weight of 100 kDa to 500 kDa.

[0455] 45. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight average molecular weight of 100 kDa to 300 kDa.

[0456] 46. ​​The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight average molecular weight of 200 kDa to 1000 kDa.

[0457] 47. The isolated polysaccharide of any one of paragraphs 1 to 39, having a weight average molecular weight of 200 kDa to 500 kDa.

[0458] 48. The isolated polysaccharide of any one of paragraphs 1 to 39, wherein the isolated capsular polysaccharide has a weight average molecular weight of 300 kDa to 400 kDa.

[0459] 49. The isolated polysaccharide of any one of paragraphs 1 to 39, wherein the isolated capsular polysaccharide has a weight average molecular weight of 100 kDa to 500 kDa.

[0460] 50. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate prepared by a process comprising: a) reacting the isolated polysaccharide of any one of paragraphs 1 to 49 with an activating agent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein.

[0461] 51. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0462] 52. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0463] 53. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0464] 54. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0465] 55. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0466] 56. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0467] 57. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0468] 58. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0469] 59. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0470] 60. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0471] 61. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0472] 62. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0473] 63. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0474] 64. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0475] 65. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0476] 66. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0477] 67. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeating units of the polysaccharide.

[0478] 68. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0479] 69. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeating units of the polysaccharide.

[0480] 70. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0481] 71. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0482] 72. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0483] 73. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0484] 74. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0485] 75. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0486] 76. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0487] 77. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeating units of the polysaccharide.

[0488] 78. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0489] 79. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0490] 80. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0491] 81. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0492] 82. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0493] 83. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeating units of the polysaccharide.

[0494] 84. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues per 100 sugar repeating units of the polysaccharide.

[0495] 85. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0496] 86. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0497] 87. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0498] 88. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0499] 89. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0500] 90. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.05 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0501] 91. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0502] 92. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0503] 93. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0504] 94. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0505] 95. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0506] 96. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0507] 97. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0508] 98. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 0.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0509] 99. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0510] 100. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0511] 101. Streptococcus pneumoniae ( S. pneumoniae ) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 sugar repeating units of the polysaccharide.

[0512] 102. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0513] 103. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0514] 104. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0515] 105. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0516] 106. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 2 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0517] 107. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0518] 108. Serotype 12F glycoconjugates, including serotype 12F capsular polysaccharides containing about 3 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0519] 109. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0520] 110. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 3 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0521] 111. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0522] 112. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0523] 113. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0524] 114. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 4 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0525] 115. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0526] 116. Streptococcus pneumoniae ( S. pneumoniae ) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0527] 117. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0528] 118. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0529] 119. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0530] 120. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0531] 121. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 sugar repeat units of the polysaccharide.

[0532] 122. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing about 10 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0533] 123. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0534] 124. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing from about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0535] 125. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0536] 126. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0537] 127. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0538] 128. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing from about 0.1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0539] 129. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0540] 130. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0541] 131. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 0.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0542] 132. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0543] 133. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 0.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0544] 134. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 0.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0545] 135. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 1 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 1 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0546] 136. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing from about 1 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 1 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0547] 137. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 1 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 1 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0548] 138. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 1 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 1 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0549] 139. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 2 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 2 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0550] 140. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 2 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 2 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0551] 141. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 2 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 2 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0552] 142. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 2 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 2 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0553] 143. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 3 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0554] 144. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 3 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0555] 145. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 3 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0556] 146. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 3 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 3 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0557] 147. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 4 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0558] 148. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 4 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0559] 149. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 4 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0560] 150. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 4 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 4 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0561] 151. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0562] 152. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0563] 153. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0564] 154. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0565] 155. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 7.5 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7.5 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0566] 156. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 7.5 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7.5 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0567] 157. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 7.5 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7.5 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0568] 158. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, which contains a serotype 12F capsular polysaccharide containing about 7.5 to about 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 7.5 to about 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0569] 159. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 10 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and about 10 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0570] 160. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 10 to about 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 10 to about 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0571] 161. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing about 10 to about 15 N-acetyl-D-fucosamine (D-FucNAc) residues and about 10 to about 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0572] 162. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 0.1 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 0.1 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0573] 163. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate, comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.05 to about 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0574] 164. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.05 to about 1 N-acetyl-D-fucosamine (D-FucNAc) residue and about 0.05 to about 1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0575] 165. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 0.2 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 0.2 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0576] 166. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 0.1 to about 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

[0577] 167. A Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugate comprising a serotype 12F capsular polysaccharide containing about 0.1 to about 1 N-acetyl-D-fucosamine (D-FucNAc) residue and about 0.1 to about 1 N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 saccharide repeating units of the polysaccharide.

[0578] 168. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing approximately 0.05 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 0.05 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0579] 169. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates containing a serotype 12F capsular polysaccharide containing approximately 0.1 N-acetyl-D-fucosamine (D-FucNAc) residue and approximately 0.1 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0580] 170. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 0.5 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 0.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0581] 171. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing approximately one N-acetyl-D-fucosamine (D-FucNAc) residue and approximately one N-acetyl-D-quinovosamine (D-QuiNAc) residue per 100 sugar repeating units of the polysaccharide.

[0582] 172. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, including a serotype 12F capsular polysaccharide containing approximately two N-acetyl-D-fucosamine (D-FucNAc) residues and approximately two N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0583] 173. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately three N-acetyl-D-fucosamine (D-FucNAc) residues and approximately three N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0584] 174. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately five N-acetyl-D-fucosamine (D-FucNAc) residues and approximately five N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0585] 175. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately seven N-acetyl-D-fucosamine (D-FucNAc) residues and approximately seven N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0586] 176. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 12 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 12 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0587] 177. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0588] 178. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 13 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 13 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0589] 179. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 14 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 14 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0590] 180. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 15 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 15 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0591] 181. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 22.5 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 22.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0592] 182. Streptococcus pneumoniae (S. pneumoniae) serotype 12F glycoconjugates, which contain a serotype 12F capsular polysaccharide containing approximately 25 N-acetyl-D-fucosamine (D-FucNAc) residues and approximately 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 sugar repeating units of the polysaccharide.

[0593] 183. The glycoconjugate of any one of paragraphs 50 to 182, comprising serotype 12F polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide before conjugation is between 50 kDa and 1,000 kDa.

[0594] 184. The glycoconjugate according to any one of paragraphs 50 to 182, comprising serotype 12F polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide before conjugation is between 100 kDa and 600 kDa.

[0595] 185. The glycoconjugate of any one of paragraphs 50 to 182, comprising serotype 12F polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide before conjugation is between 150 kDa and 400 kDa.

[0596] 186. The glycoconjugate of any one of paragraphs 50 to 185, having a weight average molecular weight (Mw) of 250 kDa to 15,000 kDa.

[0597] 187. The glycoconjugate of any one of paragraphs 50 to 185, having a weight average molecular weight (Mw) of 500 kDa to 2,500 kDa.

[0598] 188. The glycoconjugate of any one of paragraphs 50 to 185, having a weight average molecular weight (Mw) of 1,000 kDa to 2,500 kDa.

[0599] 189. The glycoconjugate according to any one of paragraphs 50 to 188, having a conjugation degree of 2 to 15.

[0600] 190. The glycoconjugate according to any one of paragraphs 50 to 188, having a conjugation degree of 2 to 10.

[0601] 191. The glycoconjugate according to any one of paragraphs 50 to 188, having a conjugation degree of 2 to 6.

[0602] 192. The glycoconjugate according to any one of paragraphs 50 to 188, having a conjugation degree of 3 to 5.

[0603] 193. The glycoconjugate according to any one of paragraphs 50 to 188, having a conjugation degree of 4 to 10.

[0604] 194. The glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio of serotype 12F polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.5 to 3.0.

[0605] 195. The glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio of serotype 12F polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.5 to 2.0.

[0606] 196. The glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio (w / w) of serotype 12F polysaccharide to carrier protein in the glycoconjugate is 0.5 to 1.5.

[0607] 197. The glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio of serotype 12F polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.8 to 1.2.

[0608] 198. The glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio of serotype 12F polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.5 to 1.0.

[0609] 199. A glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio (w / w) of serotype 12F polysaccharide to carrier protein in the glycoconjugate is 1.0 to 1.5.

[0610] 200. A glycoconjugate according to any one of paragraphs 50 to 193, wherein the ratio (w / w) of serotype 12F polysaccharide to carrier protein in the glycoconjugate is 0.9 to 1.1.

[0611] 201. The glycoconjugate of any one of paragraphs 50 to 200, comprising less than about 50% free serotype 12F polysaccharide compared to the total amount of serotype 12F polysaccharide.

[0612] 202. The glycoconjugate of any one of paragraphs 50 to 200, comprising less than about 25% free serotype 12F polysaccharide compared to the total amount of serotype 12F polysaccharide.

[0613] 203. The glycoconjugate of any one of paragraphs 50 to 200, comprising less than about 20% free serotype 12F polysaccharide compared to the total amount of serotype 12F polysaccharide.

[0614] 204. The glycoconjugate of any one of paragraphs 50 to 200, comprising less than about 15% free serotype 12F polysaccharide compared to the total amount of serotype 12F polysaccharide.

[0615] 205. At least 30% of serotype 12F glycoconjugates have a K value of 0.3 or less in a CL-4B column. d The complex carbohydrate described in any one of paragraphs 50 to 204, having the following structure:

[0616] 206. At least 60% of serotype 12F glycoconjugates have a K value of 0.3 or less in a CL-4B column. d The complex carbohydrate described in any one of paragraphs 50 to 204, having the following structure:

[0617] 207. 50% to 80% of serotype 12F glycoconjugates have a K value of 0.3 or less in the CL-4B column. d The complex carbohydrate described in any one of paragraphs 50 to 204, having the following structure:

[0618] 208. 65% to 80% of serotype 12F glycoconjugates exhibit a K value of 0.3 or less in the CL-4B column. d The complex carbohydrate described in any one of paragraphs 50 to 204, having the following structure:

[0619] 209. Carrier proteins of glycoconjugates are TT (tetanus toxoid), DT (diphtheria toxoid), DT mutant (CRM 197 209. The glycoconjugate according to any one of paragraphs 50 to 208, wherein the glycoconjugate is selected from the group consisting of C5a peptidase from Streptococcus (SCP), and the like.

[0620] 210. The glycoconjugate according to any one of paragraphs 50 to 208, wherein the carrier protein of the glycoconjugate is DT (diphtheria toxoid).

[0621] 211. The glycoconjugate according to any one of paragraphs 50 to 208, wherein the carrier protein of the glycoconjugate is TT (tetanus toxoid).

[0622] 212. The glycoconjugate according to any one of paragraphs 50 to 208, wherein the carrier protein of the glycoconjugate is PD (H. influenzae protein D).

[0623] 213. The glycoconjugate according to any one of paragraphs 50 to 208, wherein the carrier protein of the glycoconjugate is CRM197.

[0624] 214.CRM 197 214. The glycoconjugate of paragraph 213, wherein the saccharide comprises 1 to 15 of the 39 saccharides covalently linked to a lysine residue.

[0625] 215.CRM 197214. The glycoconjugate of paragraph 213, wherein the glycoconjugate comprises lysine residues covalently linked to 1 to 20 of the 39 sugars.

[0626] 216. A glycoconjugate according to any one of paragraphs 50 to 215, prepared using reductive amination.

[0627] 217. The glycoconjugate according to paragraph 216, wherein the oxidation is preceded by sizing of the polysaccharide to a target molecular weight (MW) range.

[0628] 218. A glycoconjugate according to any one of paragraphs 50 to 217, prepared by a process comprising the steps of: a) reacting a serotype 12F saccharide with a stable nitroxyl radical compound and an oxidizing agent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein.

[0629] 219. The glycoconjugate according to paragraph 218, wherein the stable nitroxyl radical compound is a molecule bearing a TEMPO or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety.

[0630] 220. The glycoconjugate according to paragraph 218, wherein the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinoxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamido)-TEMPO, 4-amino-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl.

[0631] 221. The glycoconjugate according to paragraph 218, wherein the stable nitroxyl radical compound is TEMPO.

[0632] 222. The glycoconjugate of any one of paragraphs 218-221, wherein the oxidizing agent is a molecule bearing an N-halo moiety.

[0633] 223. The glycoconjugate of any one of paragraphs 218 to 221, wherein the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.

[0634] 224. The glycoconjugate of any one of paragraphs 218 to 221, wherein the oxidizing agent is N-chlorosuccinimide.

[0635] 225. The glycoconjugate according to paragraph 218, wherein the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0636] 226. A glycoconjugate according to any one of paragraphs 218 to 225, wherein at the end of the reduction reaction, any unreacted aldehyde groups remaining in the conjugate are capped using a capping agent.

[0637] 227. The glycoconjugate according to paragraph 226, wherein the capping agent is sodium borohydride (NaBH4).

[0638] 228. The glycoconjugate of any one of paragraphs 50 to 217, prepared by a process comprising the steps of: (a) reacting isolated serotype 12F polysaccharide with an oxidizing agent; (b) combining the activated polysaccharide of step (a) with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form the glycoconjugate.

[0639] 229. The glycoconjugate of any one of paragraphs 50 to 217, prepared by a process comprising the steps of: (a) reacting isolated serotype 12 F polysaccharide with an oxidizing agent; (a') quenching the oxidation reaction by adding a quenching agent; (b) combining the activated polysaccharide of step (a') with a carrier protein; and (c) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form the glycoconjugate.

[0640] 230. The glycoconjugate of any one of paragraphs 228-229, wherein the oxidizing agent is periodate.

[0641] 231. The glycoconjugate according to any one of paragraphs 228 to 230, wherein the oxidation degree of the active serotype 12F polysaccharide is 2 to 30.

[0642] 232. The glycoconjugate according to any one of paragraphs 228 to 231, wherein at the end of the reduction reaction, any unreacted aldehyde groups remaining in the conjugate are capped using a capping agent.

[0643] 233. The glycoconjugate according to paragraph 232, wherein the capping agent is sodium borohydride (NaBH4).

[0644] 234. An immunogenic composition comprising a polysaccharide according to any one of paragraphs 1 to 49.

[0645] 235. An immunogenic composition comprising a glycoconjugate according to any one of paragraphs 50 to 234.

[0646] 236. The immunogenic composition of paragraph 235, comprising 1 to 25 glycoconjugates from different serotypes of S. pneumoniae.

[0647] 237. The immunogenic composition of paragraph 235, comprising 20 glycoconjugates from different serotypes of S. pneumoniae.

[0648] 238. The immunogenic composition of paragraph 235, which is a 20-valent pneumococcal conjugate composition.

[0649] 239. The immunogenic composition of paragraph 235, further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

[0650] 240. The immunogenic composition of paragraph 239, further comprising glycoconjugates from S. pneumoniae serotypes 1, 5, and 7F.

[0651] 241. The immunogenic composition of paragraph 240, further comprising a glycoconjugate from S. pneumoniae serotype 3.

[0652] 242. The immunogenic composition of paragraph 241, further comprising glycoconjugates from S. pneumoniae serotypes 6A and 19A.

[0653] 243. The immunogenic composition of paragraph 242, further comprising glycoconjugates from S. pneumoniae serotypes 22F and 33F.

[0654] 244. The immunogenic composition of paragraph 243, further comprising glycoconjugates from S. pneumoniae serotypes 8, 10A, 11A, and 15B.

[0655] 245. The immunogenic composition of paragraph 244, which is a 20-valent pneumococcal conjugate composition.

[0656] 246. Glycoconjugates from Streptococcus pneumoniae serotype 12F are CRMs 197 246. The immunogenic composition of any one of paragraphs 235 to 245, wherein the immunogenic composition is conjugated to

[0657] 247. Complex carbohydrates are all CRMs 197247. The immunogenic composition of any one of paragraphs 235 to 246, wherein the immunogenic composition is conjugated to

[0658] 248. The immunogenic composition of any one of paragraphs 235 to 247 for use as a medicament.

[0659] 249. The immunogenic composition of any one of paragraphs 235 to 247 for use as a vaccine.

[0660] 250. A method for detecting the presence of 4-keto-N-acetyl-quinovosamine residues in isolated S. pneumoniae serotype 12 F polysaccharide, comprising the steps of: a) isolating S. pneumoniae serotype 12 F polysaccharide; and b) detecting the presence of 4-keto-N-acetyl-quinovosamine residues in the polysaccharide.

[0661] 251. The method of paragraph 250, wherein the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by NMR or mass spectrometry (MS).

[0662] 252. The method of paragraph 250, wherein the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by NMR.

[0663] 253. The method of paragraph 250, wherein the presence of the 4-keto-N-acetyl-quinovosamine residue is detected by mass spectrometry (MS).

[0664] 254. A method for determining the amount of 4-keto-N-acetyl-quinovosamine residues in isolated S. pneumoniae serotype 12F polysaccharide, comprising the steps of: a) isolating S. pneumoniae serotype 12F polysaccharide; and b) measuring the amount of 4-keto-N-acetyl-quinovosamine residues in the polysaccharide.

[0665] 255. The method of paragraph 254, wherein the amount of 4-keto-N-acetyl-quinovosamine residue is determined by NMR.

[0666] 256. The method of paragraph 254, wherein the amount of 4-keto-N-acetyl-quinovosamine residue is determined by mass spectrometry (MS).

[0667] 257. A method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in reduced Streptococcus pneumoniae (S. pneumoniae) serotype 12 F polysaccharide, comprising the steps of: a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent; and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in the reduced polysaccharide.

[0668] 258. The method of paragraph 257, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by NMR.

[0669] 259. The method of paragraph 254, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by mass spectrometry (MS).

[0670] 260. The method of any one of paragraphs 254 to 259, wherein the reducing agent is sodium borohydride (NaBH4).

[0671] 261. The method of any one of paragraphs 254 to 260, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been previously treated with an oxidizing agent.

[0672] 262. The method of paragraph 261, wherein the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes.

[0673] 263. The method of paragraph 261, wherein the oxidizing agent is periodate.

[0674] 264. The method of any one of paragraphs 254 to 260, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with a stable nitroxyl radical compound and an oxidizing agent.

[0675] 265. The method of paragraph 263, wherein the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0676] 266. A method for detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in reduced serotype 12 F polysaccharide, comprising the steps of: a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent; and b) detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in the reduced polysaccharide.

[0677] 267. The method of paragraph 266, wherein the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0678] 268. The method of paragraph 266, wherein the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS).

[0679] 269. The method of any one of paragraphs 266-268, wherein the reducing agent is sodium borohydride (NaBH4).

[0680] 270. The method of any one of paragraphs 266-269, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been previously treated with an oxidizing agent.

[0681] 271. The method of paragraph 270, wherein the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes.

[0682] 272. The method of paragraph 270, wherein the oxidizing agent is periodate.

[0683] 273. The method of any one of paragraphs 266-269, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been pretreated with a stable nitroxyl radical compound and an oxidizing agent.

[0684] 274. The method of paragraph 273, wherein the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0685] 275. A method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in reduced Streptococcus pneumoniae (S. pneumoniae) serotype 12 F polysaccharide, comprising the steps of: a) reacting isolated S. pneumoniae serotype 12 F polysaccharide with a reducing agent; and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in the reduced polysaccharide.

[0686] 276. The method of paragraph 275, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0687] 277. The method of paragraph 275, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS).

[0688] 278. In one embodiment, the reducing agent is sodium borohydride (NaBH4).

[0689] 279. The method of any one of paragraphs 275 to 278, wherein the reducing agent is sodium borohydride (NaBH4).

[0690] 280. The method of any one of paragraphs 275-279, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been previously treated with an oxidizing agent.

[0691] 281. The method of paragraph 280, wherein the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to aldehydes.

[0692] 282. The method of paragraph 280, wherein the oxidizing agent is periodate.

[0693] 283. The method of any one of paragraphs 275-279, wherein the isolated S. pneumoniae serotype 12F polysaccharide has been previously treated with a stable nitroxyl radical compound and an oxidizing agent.

[0694] 284. The method of paragraph 283, wherein the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and the oxidizing agent is N-chlorosuccinimide (NCS).

[0695] 285. A method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of: a) preparing S. pneumoniae serotype 12F glycoconjugates; and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0696] 286. The method of paragraph 285, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0697] 287. The method of paragraph 285, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and / or N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS).

[0698] 288. A method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of: a) preparing S. pneumoniae serotype 12F glycoconjugates; and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0699] 289. The method of paragraph 288, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0700] 290. The method of paragraph 288, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) and N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS).

[0701] 291. A method for detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of: a) preparing S. pneumoniae serotype 12F glycoconjugates; and b) detecting the presence of N-acetyl-D-fucosamine (D-FucNAc) residues in said glycoconjugates.

[0702] 292. The method of paragraph 291, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by NMR.

[0703] 293. The method of paragraph 291, wherein the presence of N-acetyl-D-fucosamine (D-FucNAc) residues is detected by mass spectrometry (MS).

[0704] 294. A method for detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in S. pneumoniae serotype 12F glycoconjugates, comprising the steps of: a) preparing S. pneumoniae serotype 12F glycoconjugates; and b) detecting the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues in said glycoconjugates.

[0705] 295. The method of paragraph 294, wherein the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by NMR.

[0706] 296. The method of paragraph 294, wherein the presence of N-acetyl-D-quinovosamine (D-QuiNAc) residues is detected by mass spectrometry (MS).

[0707] As used herein, the term "about" means within a statistically significant range of values, such as a stated concentration range, time frame, molecular weight, temperature, or pH. Such a range may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% or 1% of a given value or range. In some cases, such a range may be within experimental error typical of the standard method used to measure and / or determine a given value or range. The allowable variation encompassed by the term "about" will depend on the specific system under study and will be readily understood by one of ordinary skill in the art. Whenever a range is recited herein, all numbers within the range are also contemplated as embodiments of the present disclosure.

[0708] In all cases, it is intended by the inventors that the terms "comprising," "comprise," and "comprises" herein be optionally interchangeable with the terms "consisting essentially of," "consist essentially of," "consists essentially of," "consisting of," "consist of," and "consists of," respectively.

[0709] An "immunogenic amount," "immunologically effective amount," "therapeutically effective amount," "prophylactically effective amount," or "dose," each of which is used interchangeably herein, generally refers to that amount of an antigen or immunogenic composition sufficient to elicit either a cellular (T cell) or humoral (B cell or antibody) immune response, or both, as measured by standard assays known to those skilled in the art.

[0710] Any integer within any of the ranges in this document is contemplated as an embodiment of the present disclosure.

[0711] All references or patent applications cited within this patent specification are hereby incorporated by reference.

[0712] The present invention is illustrated in the accompanying examples. The following examples are carried out using standard techniques that are well known and routine to those skilled in the art, unless otherwise specified. The examples are illustrative but do not limit the invention. [Example]

[0713] Example 1 Serotype 12F capsular polysaccharide contains 4-keto-N-acetyl-quinovosamine (4KQ) According to Leontein et al. (Leontein et al. (1981) Can. J. Chem. 59:2081-2085), the polysaccharide repeating unit of serotype 12F consists of a linear trisaccharide backbone (one N-acetylfucosamine (FucpNAc), one N-acetylgalactosamine (GalpNAc), and one N-acetylmannuronic acid (ManpNAcA)) with two branches: a pendant α-galactopyranose (Galp) linked at C3 of FucpNAc and an α-Glcp-(1→2)-α-Glcp disaccharide branch linked at C3 of ManpNAcA.

[0714] The pneumococcal polysaccharide 12F was studied by 2D NMR and mass spectrometry to characterize the polymer repeating units. It was surprisingly discovered that the serotype 12F polysaccharide indeed contains a partial replacement of N-acetyl-galactosamine with 4-keto-N-acetyl-quinovosamine (also referred to herein as 2-acetamido-2,6-dideoxy-D-xylo-4-hexulose and Sug). This ketosugar variant (4KQ) was identified as a statistical average replacement of GalNAc residues between 12F repeating units in the original strains, at approximately 20-25 mol%.

[0715] method NMR. For NMR analysis, samples were typically treated with tip sonication. 12F polysaccharide dissolved in aqueous solvent was tip-sonicated on an ice bath for up to 90 minutes. Samples were filtered using a 0.22 micron filter to remove tip particles and, in some cases, size-separated using a spin column with a fixed MWCO pore size. Sonicated samples, with or without size separation, were dialyzed against water using a 3 kDa MWCO dialysis cassette, frozen, lyophilized, and redissolved in D2O containing approximately 0.55 mM TSP-d4. The pH of the samples was approximately 6-7, adjusted with a small amount of NaOD for highly concentrated samples due to the acidity of the carboxylic acids in the backbone MannNAcA residues. NMR data were collected on a Bruker 5 mm DCH cryoprobe on a Bruker-Biospin AVANCE III NMR spectrometer operating at 500 MHz. Data processing was performed using M-Nova v12.0. Chemical shift references are for TSP-d4 at 0 and -1.8 ppm, respectively. 1 H and 13 It was C.

[0716] 1D 1 For H spectrum processing, 0.5 Hz EM line broadening was used and manual cubic spline baseline correction was applied. 13 C spectra were collected using power gating with a 0.5 second interscan recycle delay. 2D analysis was performed using 1 H- 1 H COSY, 1 H- 1 H NOESY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 13 C HSQC-TOCSY was included.

[0717] LC-MS. LC-MS and LC-MS / MS data were collected in positive ionization mode on a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with an Agilent 1260 HPLC. Samples were separated by injection onto a Waters hydrophilic interaction (HILIC) BEH spherical hybrid column. Mobile phase (MP) A was water containing 0.1% formic acid (FA), and MPB was acetonitrile (ACN) containing 0.1% FA. The elution gradient was delivered at 200 μL / min, ranging from 10 to 95% MPA over 35 min, then back to 10% MPA over 1 min and equilibrated for 15 min. The RF lens potential was increased to 70 V to induce in-source fragmentation of the polysaccharides. MS / MS data were acquired using higher-energy collisional dissociation (HCD).

[0718] SEC / MS. SEC / MS data from partial acid hydrolysis experiments were collected on a Thermo Orbitrap Elite mass spectrometer equipped with an Agilent 1260 HPLC. Samples were injected and separated onto a BEH200 SEC column. Mobile phase (MP) A was water containing 0.05% trifluoroacetic acid (TFA), and MPB was acetonitrile (ACN) containing 0.05% TFA. An isocratic gradient was delivered at 200 μL / min: 80% MPA and 20% MPB.

[0719] Partial acid hydrolysis. Native 12F polysaccharide at 6.3 mg / ml in water was treated with TFA. To an HPLC vial, 100 μl of 12F sample and 2 μl of neat TFA were added. The mixture was vortexed and placed on a hot plate set at 60°C for 100 minutes, then 70°C for 120 minutes, and then 80°C for 60 minutes. Samples were analyzed by SEC / MS on a Thermo Orbitrap Elite mass spectrometer.

[0720] result The NMR data are consistent with a single heterogeneous serotype 12F polysaccharide containing two distinct repeating unit populations. The major repeating unit population (approximately 75-80 mol%) is consistent with the constituent sugars and organization published by Leontein et al. (Leontein et al. (1981) Can. J. Chem. 59:2081-2085). The second repeating unit population (approximately 20-25 mol%) is characterized by the replacement of main-chain GalNAc residues with Sug residues (2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose), which are predominantly in the hydrate form in bulk aqueous solvent under the typical temperature and pH conditions assayed (i.e., 75°C and pH approximately 6-7). Figures 1A, 1B, and 1C illustrate the two repeating unit populations: without (Figure 1A) and with (Figure 1B) GalNAc replacement by Sug. There is no evidence of two distinct serotype 12F polysaccharides; the NMR data best fit a single serotype 12F polysaccharide with an average of 20–25% GalNAc substitutions with Sug (a keto sugar) (Fig. 1C). 1 H and / or 13 Site-specific resolved changes in the chemical shifts of C are illustrated by the shaded circles in Figure 1B.

[0721] Well-resolved shifts due to Sug residues include the CH3 at position 6 of Sug. 1 H signal, and the CH3 at position 6 of the adjacent FucNAc due to the incorporation of Sug 1 The unique feature of the Sug residue is the highly shielded CH3 at position 6 at approximately 12.36 ppm. 13 C signal with a corresponding proton chemical shift of 1.3 ppm. This is a stronger shielding than expected for this type of methyl carbon and is due to the adjacent hydrated ketone at position 4. The hydrated ketone is sp3 and has no attached proton, therefore resulting in a sharp resolved peak at approximately 94 ppm. 13The C resonance was identified by HMBC correlation as the carbon of the hydrated ketone at position 4 of the Sug residue (Figure 2). 13 The C resonance represents the serotype 12 F signal unique to the Sug residue.

[0722] Under typical analytical conditions (aqueous solvent, pH ∼6–7, 75°C), Sug ketone is predominantly in the hydrate form, and a weak signal at ∼203 ppm is attributed to Sug ketone at position 4 of the Sug residue in serotype 12F polysaccharide in an approximately 9:1 ratio (hydrate:ketone) (see Figure 3 ).

[0723] The exact molar ratio of sugar residues in the serotype 12F polysaccharide is 1D 13 The ratio of serotype 12 F polysaccharide residues was established based on the deconvolution of the C spectrum. This deconvolution technique revealed the ratio of α-L-FucNAc:α-D-Gal:β-D-GalNAc:Sug:β-D-MannNAcA:α-D-Glc (1:1:0.75:0.25:1:2), as shown in Figure 1.

[0724] The Sug residue component of serotype 12F polysaccharide is systematically present at approximately 20-25 mol% in various lots of polysaccharide from the same strain (strain 3 in Table 3 below) and is a product of fermentation.

[0725] The position of the Sug residue in the polysaccharide repeating unit is 1 H- 1 Identification is performed using H NOESY NMR spectroscopy. The NMR NOESY data show that Sug replaces GalNAc in the repeating unit (see NOESY correlation in Figure 4).

[0726] Mass spectrometry was also used to help further elucidate the structure of this unique signal in NMR. Mass spectrometry experiments were designed to generate pseudomolecular ions of the polysaccharide repeating units (RUs). By performing MS / MS experiments on the RU pseudomolecular ion at m / z 1094.3892 of the 12F polysaccharide, the complete sequence of the repeating units could be obtained.

[0727] Figure 5 is an example of repeat unit (RU) sequencing that can be achieved using mass spectrometry. In this figure, all sugar rings have been detected and identified. Figure 5 is labeled with letters A through F, representing the five sugar rings in the 12F RU. As shown in Figure 5, the linear portion of the repeat unit makes up the ring ABC. The side chain sugars are positioned on top of the sugar to which they are attached.

[0728] Figure 5 shows the structure of the 12F polysaccharide as an example of how mass spectrometry data is interpreted. The rings are labeled A-F, and the abbreviated configuration of the ring order is also shown. Molecular masses are provided in Table 1.

[0729] [Table 1]

[0730] Several experiments were performed in an attempt to determine whether the keto sugar variant of 12F was a unique polysaccharide present in the serotype 12F polysaccharide, or whether the keto sugar was part of the 12F polysaccharide that replaced GalNAc approximately 20% of the time. The first experiment was to see if the two different polysaccharides could be separated using a HILIC column. Only one polysaccharide was found that contained both GalNAc and a keto sugar.

[0731] The next experiment involved looking for dimers or trimers with all keto sugars in the polysaccharide, which would provide evidence for two different polysaccharides.

[0732] Partially hydrolyzed 12F polysaccharide was analyzed. Partial acid hydrolysis cleaves glycosidic bonds to produce species of various residue lengths. 4KQ is -18 Da relative to N-acetylgalactosamine, and dimers and trimers will yield hydrolysis products 18 Da lower if only one polysaccharide contains a keto sugar. In the case of the second species (when a chain exists in which a keto sugar replaces all N-acetylgalactosamines), dimers and trimers will yield hydrolysis products 36 Da and 54 Da lower, respectively.

[0733] The detected species are consistent with incorporation of approximately one in every four RUs having a keto sugar. This data indicates that all polysaccharide chains of 12F contain a keto sugar, with no two keto sugars in adjacent repeating units. There is no evidence of chains in which all N-acetylgalactosamines are replaced by keto sugars.

[0734] These data support the idea that 12F is a polysaccharide containing keto sugars in the repeating units at a rate of 20-25%.

[0735] Taken together, the data support that the serotype 12F polysaccharide consists of a single heterogeneous polysaccharide chain in which, on statistical average, keto sugars replace 20–25% of the N-acetylgalactosamines in the repeat unit backbone (Figure 1). There is no evidence of chains in which all N-acetylgalactosamines are replaced by keto sugars.

[0736] Example 2 Structure of reduced serotype 12F capsular polysaccharide method Reduced 12F polysaccharide. Reduced 12F polysaccharide was produced as follows: 150 mg of hydrolyzed 12F (2.5 mg / mL) in 60 mL of water, pH 7.0, was mixed with 2 mL of NaBH4 solution (approximately 807 mM in water) at 150 rpm overnight at 23 °C, and then dialyzed against water using a 7 KDa MWCO dialysis cassette. 1 For H spectrum processing, 0.5 Hz EM line broadening was used and manual cubic spline baseline correction was applied.13 C spectra were collected using power gating with a 0.5 second interscan recycle delay. 2D analysis was performed using 1 H- 1 H COSY, 1 H- 1 H NOESY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 13 C HSQC-TOCSY was included.

[0737] LC-MS and LC-MS / MS. LC-MS and LC-MS / MS data from the reduction of 12F with NaBD4 were collected in positive ionization mode on a Thermo Orbitrap Q Exactive mass spectrometer equipped with an Agilent 1260 HPLC. Samples were injected and separated onto a Waters hydrophilic interaction (HILIC) BEH spherical hybrid column. Mobile phase (MP) A was water with 0.1% TFA, and MPB was acetonitrile (ACN) with 0.1% TFA. The elution gradient was delivered at 200 μL / min, ranging from 30 to 70% MPA over 35 min, returning to 30% MPA over 1 min, and equilibrating for 14 min. The RF lens potential was increased to 60 V to induce in-source fragmentation of the polysaccharide. MS / MS data were acquired using higher-energy collisional dissociation (HCD).

[0738] Reaction with NaBD4: 6.7 mg / ml NaBD4 was dissolved in water. 0.03 ml of NaBD4 was added to 0.27 ml of 12F polysaccharide (approximately 3 mg / ml in water) and incubated for 3 hours at room temperature. 1 μl of neat acetic acid was then added to quench the reaction.

[0739] result The ketone / hydrate form of the Sug residue is sensitive to reduction using NaBH4. Serotype 12F polysaccharide treated with NaBH4 is characterized by specific changes in the Sug residue, including deshielding of the methyl carbon at position 6, loss of the sp3 carbon at approximately 94 ppm without a directly bound proton, and the appearance of two weaker novel spin systems consistent with D-FucNAc and D-QuiNAc (Figure 6). Upon reduction, the main serotype 12F polysaccharide spin system remains unchanged (Figure 1A), and the observed heterogeneity pattern of residues adjacent to the incorporation site remains the same.

[0740] Treatment of serotype 12F polysaccharide with NaBH4 specifically reduced position 4 of the Sug residue from a ketone / hydrate to an alcohol, converting the residue Sug to a mixture of D-FucNAc and D-QuiNAc, characterized by the hydroxyl at position 4 in the axial and equatorial orientations, respectively, as illustrated in Figure 6.

[0741] After reduction with NaBD4, the masses of RU and fragment ions containing keto sugar residues are shifted higher than 3.0219 Da. This atypical mass shift from deuterium is unnatural. 12 C. 13 C. 1 H, 16 O, and 14 It provides a unique ion with no interference from fragment ions containing natural isotopes of N. This data supports the reduction of ketones with NaBD4 (see Table 2 and Figure 7).

[0742] [Table 2]

[0743] Example 3 Levels of 4KQ substitution among circulating clinical 12F isolates. method Production, purification, and analysis of 12F polysaccharide. Culture stocks as frozen cell suspensions were prepared by growing the strain to late logarithmic phase in soy hydrolysate medium and freezing at -70°C. Production of 12F polysaccharide in fermentation medium was carried out by first creating a seed culture from the frozen stock in soy hydrolysate medium. The starter culture was then used to inoculate the same culture medium, and fermentation was carried out in a stirred bioreactor at 36°C. The broth was then dissolved by adding N-lauryl sulfonate to 0.1% and subjected to the purification procedure.

[0744] Polysaccharide purification was carried out as described in WO2020 / 170190.

[0745] result To determine whether the 4KQ substitution was common among circulating clinical isolates, 17 confirmed 12F clinical isolates were analyzed. All 17 of the 12F clinical isolates had evidence of this 4KQ substitution, ranging from 2.5 to 10.2% of the repeat units (Table 3).

[0746] Four cultures available at ATCC (American Type Culture Collection (ATCC), Manassas, VA, USA, reference number 196-X) and purified 12F pneumococcal polysaccharide were also analyzed (Table 3 ).

[0747] The amount of 4-KQ substitution was quantified by 2D-NMR (see Example 1). The % keto substitution in Pn-12F is determined using the ratio of the intensities of the methyl peaks corresponding to 4KQ to FucNAc.

[0748] The level of 4KQ substitution that occurs during fermentation depends on the strain used (see Table 3).

[0749] [Table 3]

[0750] Example 4 Immunogenicity of 4KQ-containing 12F polysaccharide complexes in OPA using clinical isolates with various levels of 4KQ method Opsonophagocytosis assay (OPA) of S. pneumoniae microcolonies. The opsonophagocytosis assay was performed as described (see, e.g., WO2018 / 134693). This assay quantitatively assesses functional anti-S. pneumoniae antibodies by measuring bacterial killing in reactions containing serially diluted test serum, baby rabbit complement, and differentiated effector cells (HL-60). The OPA titer is the reciprocal of the serum dilution that results in a 50% reduction in the number of bacterial colony-forming units (CFU) compared to a no-serum control (defined as background CFU). Titers are interpolated from the two dilutions that encompass this 50% kill cutoff. Titers from multiple determinations per sample are reported as geometric mean titers (GMT).

[0751] result To determine whether the 4KQ content of capsular polysaccharide affects the ability of vaccine-induced antibodies to bind to and kill S. pneumoniae serotype 12F isolates, the immunogenicity of multivalent vaccines containing 12F conjugates and plain 12F polysaccharide was evaluated against a set of S. pneumoniae 12F isolates with varying levels of 4KQ modification. OPA assays were performed on six 12F isolates with varying levels of 4KQ modification, and titers were generated for serum sets from subjects immunized with the multivalent vaccine containing the 12F conjugate (n = 41 individuals), the multivalent vaccine containing plain 12F polysaccharide (n = 26 individuals), or the multivalent vaccine that did not contain 12F polysaccharide (n = 28 individuals, as a negative control). The 12F polysaccharide included in the multivalent vaccine containing plain 12F polysaccharide has very low 4KQ incorporation (approximately 0.2%), while the 12F polysaccharide used in the multivalent vaccine containing the 12F conjugate contains a 4KQ modification level of approximately 25%.

[0752] As shown in Figure 8, sera immune to both the 12F conjugate-containing vaccine (12F conj.) and the plain 12F polysaccharide-containing vaccine (12F plain) were able to elicit bacterial killing responses of isolates with 4KQ modification levels ranging from 1.9% to 27.5%, with no statistically significant differences between titers. These data indicate that the vaccine elicits similar OPA titers across strains expressing low to high levels of 4KQ modification.

[0753] All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0754] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims. [Accession number]

[0755] ATCC 53281 ATCC 196-X

Claims

1. An isolated polysaccharide having the following repeating units: 【Chemistry 1】 wherein n represents the number of repeating units and X represents either N-acetylgalactosamine, N-acetyl-D-fucosamine (D-FucNAc) or N-acetyl-D-quinovosamine (D-QuiNAc), wherein the polysaccharide comprises from about 99.9 to about 50 N-acetylgalactosamine residues, from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and from about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues for every 100 saccharide repeating units of the polysaccharide.

2. A S. pneumoniae serotype 12F glycoconjugate comprising an S. pneumoniae serotype 12F capsular polysaccharide comprising from about 0.05 to about 25 N-acetyl-D-fucosamine (D-FucNAc) residues and / or from about 0.05 to about 25 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeating units of the polysaccharide.

3. 3. The polysaccharide complex of claim 2, wherein the serotype 12F capsular polysaccharide comprises about 1 to 12.5 N-acetyl-D-fucosamine (D-FucNAc) residues and about 1 to 12.5 N-acetyl-D-quinovosamine (D-QuiNAc) residues per 100 saccharide repeat units of the polysaccharide.

4. The complex carbohydrate according to claim 2 or 3, wherein the weight-average molecular weight (Mw) of the polysaccharide is 50 kDa to 1,000 kDa.

5. 5. The glycoconjugate according to any one of claims 2 to 4, wherein the ratio of polysaccharide to carrier protein (w / w) in the glycoconjugate is from 0.5 to 3.

0.

6. The glycoconjugate carrier protein is TT (tetanus toxoid), DT (diphtheria toxoid), DT mutant (CRM １９７ 6. The glycoconjugate according to claim 2, wherein the glycoconjugate is a C5a peptidase (SCP) from Streptococcus, or a C5a peptidase from Streptococcus (SCP).

7. Carrier proteins of complex carbohydrates are CRM １９７ The complex carbohydrate according to any one of claims 2 to 5,

8. CRM １９７ 8. The glycoconjugate of claim 7, wherein comprises lysine residues covalently linked to 1 to 15 of the 39 sugars.

9. 9. An immunogenic composition comprising a polysaccharide according to claim 1 or a glycoconjugate according to any one of claims 2 to 8.

10. The immunogenic composition of claim 9 for use as a medicament.

11. The immunogenic composition of claim 9 for use as a vaccine.

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