RU2827429C1 - Immunogenic compositions for use in pneumococcal vaccines - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a method of inducing an immune response in a human subject against S. pneumoniae serotypes 15B and 15C, comprising administering to said subject an immunogenic composition containing an immunogenically effective amount of a glycoconjugate containing a carrier protein and capsular saccharide from serotype 15B S. pneumoniae, wherein said composition does not contain capsular saccharide from serotype 15C S. pneumoniae.

EFFECT: invention extends the range of agents for inducing an immune response in a human subject against Streptococcus pneumoniae serotypes 15B and 15C.

15 cl, 1 dwg, 2 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention aims to provide immunogenic compositions for protection against S. pneumoniae serogroup 15. The immunogenic compositions of the present invention typically comprise conjugated capsular saccharide antigens (glycoconjugates), wherein the saccharides are derived from serotypes of Streptococcus pneumoniae. The present invention relates to novel immunogenic compositions for use in pneumococcal vaccines.

LEVEL OF TECHNOLOGY

Infections caused by pneumococci are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, while dissemination of bacteria to the respiratory tract may result in middle ear infection, sinusitis, or recurrent bronchitis. Compared with invasive disease, noninvasive manifestations are usually less severe but are much more common.

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

Pneumococcal conjugate vaccines (PCVs) are pneumococcal vaccines used to protect against disease caused by S. pneumoniae (pneumococcus). There are currently three PCV vaccines available globally: PREVNAR® (called Prevenar in some countries) (a heptavalent vaccine), SYNBLORIX® (a decavalent vaccine), and PREVNAR 13® (a tridecavalent vaccine).

Specific serotypes causing disease, other than the 13 in PREVNAR 13®, vary by region, population, and may change over time due to the acquisition of antibiotic resistance, the introduction of pneumococcal vaccine, and long-term trends of unknown origin.

Addition of conjugates to an immunogenic composition is not a simple process, since combining conjugates into a single multivalent injection may result in competition between the different components and may adversely affect the immunogenicity of any single conjugate.

This interference phenomenon may limit the number of conjugates that can be included in a multivalent vaccine. Therefore, obtaining protection against a large number of serotypes while limiting the number of conjugates in the formulation may be difficult, despite the considerable value.

S. pneumoniae serogroup 15 includes four serotypes: 15A, 15B, 15C, and 15F.

Serotypes 15A, 15B, and 15C are of particular concern, while 15F appears to pose less of a medical risk (see, e.g., Cui YA et al. Hum Vaccin Immunother. 2017; 13(6):1–13).

The aim of the present invention is to provide immunogenic compositions or a vaccine administration regimen to provide adequate protection against S. pneumoniae, in particular against S. pneumoniae serotypes 15A, 15B and 15C, while limiting the number of conjugates.

ESSENCE OF THE INVENTION

WO 2019/139692 tested serogroup 15 cross-protection (see, for example, Example 42, pages 133-134). WO 2019/139692 showed that immunization with monovalent pneumococcal conjugate vaccines of serogroups 15A, 15B, or 15C induced equivalent post-dose IgG and OPA titers for the homologous and heterologous polysaccharide and bacterial strain, respectively. WO 2019/139692 showed that rabbits immunized with conjugate 15A, conjugate 15B, or conjugate 15C were cross-reactive to each pneumococcal polysaccharide (15A, 15B, and 15C) (see Fig. 17). WO 2019/139692 concluded that there was no significant difference in IgG titers in serogroup 15 and no cross-reactivity to each bacterial strain of S. pneumoniae (15A, 15B, 15C) in all rabbits immunized with the 15A, 15B or 15C conjugate, since all hyperimmune sera from rabbits contained functional antibodies to each strain evaluated and killed bacteria (see Fig. 18 and p. 134, lines 9-13). WO 2019/139692 found no significant difference in OPA titers in serogroup 15.

Contrary to what is proposed in WO 2019/139692, it was unexpectedly found that in humans, although the serotype 15B polysaccharide conjugate, in addition to generating functional reactive antibodies to serogroup 15B, can additionally induce a strong cross-functional response against serotype 15C isolates, it induces only a minor cross-functional response against serotype 15A.

Therefore, to achieve optimal protection against S. pneumoniae serotypes 15A, 15B, and 15C, it is preferable to immunize the subject with both the serotype 15B polysaccharide conjugate and the serotype 15A polysaccharide conjugate, or with both the serotype 15C polysaccharide conjugate and the serotype 15A polysaccharide conjugate.

To achieve protection against S. pneumoniae serotypes 15A, 15B, and 15C, the subject can also be immunized with serotype 15B polysaccharide conjugate, serotype 15C polysaccharide conjugate, and serotype 15A polysaccharide conjugate.

The present invention relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not contain a capsular saccharide of S. pneumoniae serotype 15C when the glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not contain a capsular saccharide of S. pneumoniae serotype 15B when the glycoconjugate of S. pneumoniae serotype 15C is present in the composition.

The present invention relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not contain a capsular saccharide of S. pneumoniae serotype 15C.

The present invention also relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not contain a capsular saccharide of S. pneumoniae serotype 15B.

The present invention also relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C.

In one aspect, the invention relates to a set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15B, for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15C, for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, for use in a method of immunizing a human against infection with S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a method of immunizing a human subject against an infection with S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to a human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C when a glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not comprise a capsular saccharide of S. pneumoniae serotype 15B when a glycoconjugate of S. pneumoniae serotype 15C is present.

In one aspect, the invention relates to a method of immunizing a human subject against an infection with S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to the human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C.

In one aspect, the invention relates to a method of immunizing a human subject against an infection with S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to the human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15B.

In one aspect, the invention relates to a method of immunizing a human subject against infection with S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to the human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A.

The present invention relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A for use in a method for preventing, treating or alleviating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not contain a capsular saccharide of S. pneumoniae serotype 15C when the glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not contain a capsular saccharide of S. pneumoniae serotype 15B when the glycoconjugate of S. pneumoniae serotype 15C is present in the composition.

The present invention relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A for use in a method for preventing, treating or alleviating in a human an infection, disease or condition associated with an infection caused by S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not contain a capsular saccharide of S. pneumoniae serotype 15C.

The present invention also relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A for use in a method for preventing, treating or alleviating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C. Said composition preferably does not comprise a capsular saccharide of S. pneumoniae serotype 15B.

The present invention also relates to an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A for use in a method for preventing, treating or alleviating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C.

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15B, for use in a method of preventing, treating or ameliorating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15C, for use in a method of preventing, treating or ameliorating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A, for use in a method of preventing, treating or ameliorating in a human an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

In one aspect, the invention relates to a method for preventing, treating or ameliorating an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to a human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C when a glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not comprise a capsular saccharide of S. pneumoniae serotype 15B when a glycoconjugate of S. pneumoniae serotype 15C is present.

In one aspect, the invention relates to a method for preventing, treating or alleviating an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to a human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C.

In one aspect, the invention relates to a method for preventing, treating or alleviating an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to a human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A, wherein said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15B.

In one aspect, the invention relates to a method for preventing, treating or ameliorating an infection, disease or condition associated with an infection by S. pneumoniae serotypes 15A, 15B and 15C, wherein said method comprises administering to a human subject an immunologically effective amount of an immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 4, 6B, 9V, 14, 19F and/or 23F.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 4, 6B, 9V, 14, 18C, 19F and/or 23F.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 1, 5 and/or 7F.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 6A and/or 19A.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 3, 15B, 22F, 33F, 12F, 10A, 11A and/or 8.

In another aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 2, 15C, 17F and/or 20.

In one aspect, the above immunogenic compositions or set of immunogenic compositions further comprise at least one glycoconjugate of S. pneumoniae serotype 9N.

In another aspect, the immunogenic compositions or set of immunogenic compositions are a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-valent pneumococcal conjugate composition.

In another aspect, the glycoconjugates of the immunogenic compositions or set of immunogenic compositions are individually conjugated to CRM ₁₉₇ .

In one aspect, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7B, 9V, 14 and/or 23F, if present in the immunogenic compositions, are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C, if present, is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F, if present, is conjugated to DT.

In one aspect, the glycoconjugates are prepared using reductive amination chemistry. In one aspect, the glycoconjugates are prepared using CDAP chemistry.

The immunogenic compositions or set of immunogenic compositions may further comprise antigens of other pathogens and/or at least one adjuvant such as aluminum phosphate, aluminum sulfate or aluminum hydroxide.

In one aspect, the immunogenic compositions or set of immunogenic compositions are capable of inducing in a human IgG antibodies that are capable of binding to the polysaccharide of S. pneumoniae serotypes 15A, 15B and 15C at a concentration of at least 0.35 μg/mL, as determined by an ELISA assay.

In one aspect, the immunogenic compositions or set of immunogenic compositions are capable of inducing a titer of at least 1:8 against S. pneumoniae serotypes 15A, 15B, and 15C in at least 50% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA).

In one aspect, the immunogenic compositions or a set of immunogenic compositions are capable of significantly increasing the proportion of responders against S. pneumoniae serotypes 15A, 15B, and 15C compared to a pre-immunized population.

In one aspect, the immunogenic compositions or a set of immunogenic compositions are capable of significantly increasing OPA titers in humans against S. pneumoniae serotypes 15A, 15B, and 15C compared to a pre-immunized population.

In one aspect, the present invention relates to the use of an immunogenic composition disclosed herein for the preparation of a medicament for the prevention, treatment or amelioration in a subject of an infection, disease or condition caused by S. pneumoniae serotypes 15A, 15B and 15C, for use in preventing an infection with S. pneumoniae serotypes 15A, 15B and 15C in a subject, or for use in a method of protecting or treating a human susceptible to an infection with S. pneumoniae serotypes 15A, 15B and 15C, by administering said immunogenic compositions systemically or mucosally.

The invention also relates to a kit comprising an immunogenic composition or a set of immunogenic compositions disclosed in the present description and an information leaflet indicating the ability of the composition or set of immunogenic compositions to induce functional antibodies against S. pneumoniae serotypes 15A, 15B and 15C.

DESCRIPTION OF DRAWINGS

Fig. 1. Inverse cumulative distribution curves of OPA titers to serotype 15C from a panel of sera obtained from vaccinated individuals who were administered 20vPnC containing capsular polysaccharide 15B conjugate or 13vPnC without capsular polysaccharide 15B conjugate (see Example 3) (study B7471002 (ClinicalTrials.gov). Identifier: NCT03313037)). Graphs represent the percentage of sera with a positive OPA titer (i.e., ≥1:8).

1. Immunogenic compositions containing a glycoconjugate of S. pneumoniae serotypes 15B and/or 15C and a glycoconjugate of S. pneumoniae serotype 15A according to the invention

The immunogenic compositions of the present invention typically comprise conjugated capsular saccharide antigens (also referred to as glycoconjugates), wherein the saccharides are derived from S. pneumoniae serotypes.

The number of capsular saccharides of S. pneumoniae may preferably vary from 2 serotypes (or "v", valency) to 25 different serotypes (25v). In one embodiment, 2 different serotypes are present. In one embodiment, 3 different serotypes are present. In one embodiment, 4 different serotypes are present. In one embodiment, 5 different serotypes are present. In one embodiment, 6 different serotypes are present. In one embodiment, 7 different serotypes are present. In one embodiment, 8 different serotypes are present. In one embodiment, 9 different serotypes are present. In one embodiment, 10 different serotypes are present. In one embodiment, 11 different serotypes are present. In one embodiment, 12 different serotypes are present. In one embodiment, 13 different serotypes are present. In one embodiment, 14 different serotypes are present. In one embodiment, 15 different serotypes are present. In one embodiment, 16 different serotypes are present. In one embodiment, 17 different serotypes are present. In one embodiment, 18 different serotypes are present. In one embodiment, 19 different serotypes are present. In one embodiment, 20 different serotypes are present. In one embodiment, 21 different serotypes are present. In one embodiment, 22 different serotypes are present. In one embodiment, 23 different serotypes are present. In one embodiment, 24 different serotypes are present. In one embodiment, 25 different serotypes are present. The capsular saccharides are conjugated to a carrier protein to form glycoconjugates as described below.

If the carrier protein is the same for 2 or more saccharides in the composition, the saccharides may be conjugated to the same carrier protein molecule (the carrier molecules having 2 or more different saccharides conjugated to it) [see, for example, WO 2004/083251].

However, in a preferred embodiment, each of the saccharides is individually conjugated to a different carrier protein molecule (each carrier protein molecule has only one type of saccharide conjugated to it). In this embodiment, the capsular saccharides are considered to be individually conjugated to the carrier protein.

For the purposes of the invention, the term "glycoconjugate" means a capsular saccharide covalently linked to a carrier protein. In one embodiment, the capsular saccharide is directly linked to the carrier protein. In another embodiment, the bacterial saccharide is linked to the protein via a spacer/linker.

1.1. Capsular saccharide according to the invention

The term "saccharide" as used herein may mean a polysaccharide or an oligosaccharide and includes both. In frequent embodiments, the saccharide is a polysaccharide, particularly the capsular polysaccharide of S. pneumoniae.

Capsular polysaccharides are obtained by standard methods known to those skilled in the art.

Typically, capsular polysaccharides are prepared by growing each serotype of S. pneumoniae in a medium (e.g., a soy-based medium), then obtaining the polysaccharides from the bacterial culture. The bacterial strains of S. pneumoniae used to obtain the corresponding polysaccharides used in the glycoconjugates of the invention may be obtained from official culture collections or clinical samples.

The organism population (of each S. pneumoniae serotype) is often scaled up from a seed vial into seed bottles and passed through one or more seed fermentors of increasing volume until production scale fermentation volumes are achieved. At the end of the growth cycle, the cells are lysed and the lysate broth is then collected for subsequent (purification) processing (see, e.g., WO 2006/110381, WO 2008/118752, and U.S. Patent Application Publication Nos. 2006/0228380), 2006/0228381, 2008/0102498, and 2008/0286838).

Individual polysaccharides are typically purified by centrifugation, precipitation, ultrafiltration and/or column chromatography (see, for example, WO 2006/110352 and WO 2008/118752).

The purified polysaccharides may be activated (e.g., chemically activated) to render them reactive (e.g., with a carrier protein, either directly or via a linker such as an eTEC spacer) so that they can then be incorporated into the glycoconjugates of the invention, as further described herein.

The capsular polysaccharides of S. pneumoniae contain repeating oligosaccharide units that can contain up to 8 sugar residues.

In one embodiment, the capsular saccharide of the invention may be a single oligosaccharide unit or a shorter than native saccharide chain of repeating oligosaccharide units. In one embodiment, the capsular saccharide of the invention is a single repeating oligosaccharide unit of the corresponding serotype.

In one embodiment, the capsular saccharide of the invention may be an oligosaccharide.

Oligosaccharides have a small number of repeating units (usually 5-15 repeating units) and are usually produced synthetically or by hydrolysis of polysaccharides.

In one embodiment, all of the capsular saccharides of the present invention and the immunogenic compositions of the present invention are polysaccharides.

High molecular weight capsular polysaccharides are capable of inducing certain immune responses in the form of antibodies due to epitopes present on the antigen surface. For use in the conjugates, compositions and methods of the present invention, isolation and purification are preferably provided

high molecular weight capsular polysaccharides.

In some embodiments, the purified polysaccharides have a molecular weight ranging from 5 kDa to 4000 kDa before conjugation. In other such embodiments, the polysaccharide has a molecular weight of 10 kDa to 4000 kDa; from 50 kDa to 4000 kDa; from 50 kDa to 3000 kDa; from 50 kDa to 2000 kDa; from 50 kDa to 1500 kDa; from 50 kDa to 1000 kDa; from 50 kDa to 750 kDa; from 50 kDa to 500 kDa; from 100 kDa to 4000 kDa; from 100 kDa to 3000 kDa; 100 kDa and 2000 kDa; from 100 kDa to 1500 kDa; from 100 kDa to 1000 kDa; from 100 kDa to 750 kDa; from 100 kDa to 500 kDa; from 100 to 400 kDa; from 200 kDa to 4000 kDa; from 200 kDa to 3000 kDa; from 200 kDa to 2000 kDa; from 200 kDa to 1500 kDa; from 200 kDa to 1000 kDa. In one embodiment, the capsular polysaccharide has a molecular weight of 200 kDa to 500 kDa. In another embodiment, the capsular polysaccharide has a molecular weight of from 100 kDa to 500 kDa.

In additional embodiments, the capsular polysaccharide has a molecular weight of 5 kDa to 100 kDa; from 7 kDa to 100 kDa; from 10 kDa to 100 kDa; from 20 kDa to 100 kDa; from 30 kDa to 100 kDa; from 40 kDa to 100 kDa; from 50 kDa to 100 kDa; from 60 kDa to 100 kDa; from 70 kDa to 100 kDa; from 80 kDa to 100 kDa; from 90 kDa to 100 kDa; from 5 kDa to 90 kDa; from 5 kDa to 80 kDa; from 5 kDa to 70 kDa; from 5 kDa to 60 kDa; from 5 kDa to 50 kDa; from 5 kDa to 40 kDa; from 5 kDa to 30 kDa; from 5 kDa to 20 kDa or from 5 kDa to 10 kDa. Any integer in any of the above ranges is considered as an embodiment of the invention.

The polysaccharide size may be slightly reduced during conventional purification procedures. In addition, the polysaccharide may be subjected to size optimization procedures prior to conjugation. Size optimization may be accomplished by mechanical or chemical treatment.

For example, serotype 15B saccharides may be mechanically size optimized (see, for example, WO 2015/110942). Serotype 15A saccharide may be mechanically size optimized (see, for example, WO 2019/139692, examples 15-16). Serotype 15C saccharide may be mechanically size optimized (see, for example, WO 2019/139692, examples 18-19).

Serotype 15A saccharide may also be chemically size optimized. Serotype 15C saccharide may also be chemically size optimized. Chemical hydrolysis may be carried out using acetic acid. Serotype 15B saccharide is preferably not chemically size optimized in order to maintain an O-acetyl content close to that found in the natural capsular polysaccharide.

Fragmentation by high-pressure homogenization can be used as mechanical size optimization. The molecular weight ranges given above refer to purified polysaccharides before conjugation (e.g. before activation).

In a preferred embodiment, the purified polysaccharides are capsular polysaccharides of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F, wherein the capsular polysaccharide has a molecular weight falling within one of the above molecular weight ranges.

In another preferred embodiment, the purified polysaccharides are capsular polysaccharides of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23B, 23F, 24F, 33F or 35B, wherein the capsular polysaccharide has a molecular weight falling within one of the above molecular weight ranges.

In the context of the present description, the term "molecular weight" of a polysaccharide or a carrier protein-polysaccharide conjugate refers to the weight average molecular weight (Mw), which can be measured by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering (MALLS) detector.

In some embodiments, the pneumococcal saccharides of serotypes 9V, 18C, 11A, 15B, 22F and/or 33F of the invention are O-acetylated. In some embodiments, the pneumococcal saccharides of serotypes 9V, 11A, 15B, 22F and/or 33F of the invention are O-acetylated. In a preferred embodiment, the pneumococcal saccharide of serotype 18C of the invention is de-O-acetylated. For example, serotype 18C saccharides can be de-O-acetylated by acid treatment (see, for example, WO 2006/110381, page 37, lines 1-4).

The degree of O-acetylation of a polysaccharide can be determined by any method known in the art, for example by proton NMR (see, for example, Lemercinier et al. (1996) Carbohydrate Research 296:83-96, Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247, WO 2005/033148 and WO 00/56357). Another commonly used method is described in Hestrin (1949) J. Biol. Chem. 180:249-261. The presence of O-acetyl groups is preferably determined by ion HPLC analysis.

The purified polysaccharides described herein are chemically activated to make them reactive with a carrier protein. These pneumococcal conjugates are prepared in separate processes and formulated into a single dosage form as described below.

1.2 Glycoconjugates according to the invention

The purified saccharides are chemically activated to make them reactive with a carrier protein (i.e., activated saccharides), either directly or via a linker. After activation, each capsular saccharide is individually conjugated to the carrier protein to form a glycoconjugate. In one embodiment, each capsular saccharide is conjugated to the same carrier protein. Chemical activation of the saccharides and subsequent conjugation to the carrier protein can be achieved by the activation and conjugation methods described herein.

S.pneumoniae capsular polysaccharides were prepared as described above.

In one embodiment, the polysaccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then linked directly or via a spacer (linker) group to an amino group on a carrier protein (preferably CRM ₁₉₇ ). For example, the spacer can be cystamine or cysteamine to form a thiolated polysaccharide that can be linked to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide (GMBS) ester) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA) or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derived saccharide is conjugated to a carrier protein (e.g. CRM ₁₉₇ ) by carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the carrier protein. Such conjugates are described, for example, in WO 93/15760, WO 95/08348 and WO 96/129094.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F are produced using CDAP chemistry. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F are produced using CDAP chemistry. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, and 23F are produced using CDAP chemistry. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are produced using CDAP chemistry. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are produced using CDAP chemistry. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are produced using CDAP chemistry.

Other suitable conjugation methods include carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of these are described in International Patent Application Publication No. WO 98/42721. Conjugation may involve a carbonyl linker, which may be formed by reaction of the free hydroxyl group of the saccharide with CDI (see Bethell et al. (1979) 1. Biol. Chern. 254:2572-257 4; Hearn et al. (1981). J. Chromatogr., 218:509-518) followed by reaction with the protein to form a carbamate linkage. This reaction may involve reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a carbamate intermediate of CDI, and coupling of the carbamate intermediate of CDI to an amino group on the protein.

In a preferred embodiment, at least one of the capsular polysaccharides of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F is conjugated to a carrier protein via reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340, and 2007/0184071, WO 2006/110381, WO 2008/079653 and WO 2008/143709).

In another preferred embodiment, at least one of the capsular polysaccharides of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23B, 23F, 24F, 33F and 35B is conjugated to a carrier protein via reductive amination.

In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 15A is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 15B is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 15C is obtained by reductive amination.

In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 18C is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 6A is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotype 19A is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugate of S. pneumoniae serotypes 3 is obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugates of S. pneumoniae serotypes 6A and 19A are obtained by reductive amination. In a preferred embodiment of the present invention, the glycoconjugates of S. pneumoniae serotypes 3, 6A and 19A are obtained by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are obtained by reductive amination. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 6B, 9V, 14, 18C, 19F and 23F are obtained by reductive amination. In a preferred embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F are obtained by reductive amination. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F are produced by reductive amination. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F are produced by reductive amination. In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are produced by reductive amination. In a preferred embodiment of the present invention, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are obtained by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F are obtained by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, 22F and 23F are obtained by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, and 23F are obtained by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are obtained by reductive amination.

In another embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F, and 33F are produced by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15A, 15B, 18C, 19A, 19F, 22F, and 23F are obtained by reductive amination.

In another preferred embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, and 23F are obtained by reductive amination.

In another embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F are produced by reductive amination.

In another embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are produced by reductive amination.

In another embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are produced by reductive amination.

In another embodiment, all glycoconjugates of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23B, 23F, 24F, 33F, and 35B are produced by reductive amination.

Reductive amination involves two steps: (1) oxidation of the polysaccharide, (2) reduction of the activated polysaccharide and carrier protein to form a conjugate. The polysaccharide is optionally hydrolyzed before oxidation. Mechanical or chemical hydrolysis can be used. Chemical hydrolysis can be carried out with acetic acid.

The oxidation step may include a reaction with periodate. For the purposes of ^the present invention, the term "periodate" includes periodate and periodic acid; the term also includes both metaperiodate ( _IO4- ⁾ and orthoperiodate ( _IO65- ), as well as various periodate salts (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate ( _NaIO4 ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidizing agent for the selective oxidation of primary hydroxyls (as described in WO 2014/097099). In said reaction, the actual oxidizing agent in the catalytic cycle is an N-oxoammonium salt. In one aspect, said stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, said stable nitroxyl or nitroxide radical compound comprises a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, said stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, said oxidizing agent is a molecule comprising an N-halogen moiety. In one aspect, said 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 aspect, said oxidizing agent is a molecule comprising an N-halogen moiety. In one aspect, said 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, said oxidizing agent is N-chlorosuccinimide.

In a preferred embodiment, the capsular polysaccharides of S. pneumoniae serotype 12F are conjugated to a carrier protein by reductive amination, wherein the oxidizing agent is the free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) is used as a co-oxidant (as described in WO 2014/097099). Therefore, in one aspect, the glycoconjugates of S. pneumoniae serotype 12F can be prepared by a method comprising the following steps: a) reacting a 12F saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to obtain an activated saccharide; and b) reacting the activated saccharide with a carrier protein containing one or more amino groups (hereinafter referred to as “TEMPO/NCS reductive amination”).

Optionally, the oxidation reaction is quenched by adding a quenching agent. The quenching agent can be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid (e.g. glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

After the polysaccharide oxidation step, the polysaccharide is considered activated and is referred to hereinafter as the "activated polysaccharide". The activated polysaccharide and the carrier protein can be lyophilized (subjected to freeze drying) either independently of each other (separate lyophilization) or together (co-lyophilization). In one embodiment, the activated polysaccharide and the carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and the carrier protein are lyophilized independently of each other.

In one embodiment, lyophilization is carried out in the presence of a non-reducing sugar, wherein suitable non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinitol.

The second step of the conjugation process is the reduction of the activated polysaccharide and the carrier protein to form the conjugate (so-called reductive amination) using a reducing agent. Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride, sodium triacetoxyborohydride, or sodium or zinc borohydride in the presence of Bronsted or Lewis acids), aminoboranes such as pyridineborane, 2-picolineborane, 2,6-diborane-methanol, dimethylamineborane, t-BuMe ⁱ PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-methylpyridineborane (PEMB), or a borohydride exchange resin. In one embodiment, the reducing agent is sodium cyanoborohydride.

In one embodiment, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HE PES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HERB, at a pH of 6.0 to 8.5, 7.0 to 8.0 or 7.0 to 7.5); in another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) solvent or in DMF (dimethylformamide). DMSO or DMF solvent can be used to reconstitute the activated polysaccharide and carrier protein that have been lyophilized.

At the end of the reduction reaction, the conjugates may have unreacted aldehyde groups that can be blocked with a suitable blocking agent. In one embodiment, the blocking agent is sodium borohydride ( _NaBH4 ). After conjugation (reduction reaction and, optionally, copying), the glycoconjugates can be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by various methods known to those skilled in the art. These methods include dialysis, concentration/diafiltration operations, precipitation/eluting in tangential flow filtration mode, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration. In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

In one embodiment, the glycoconjugates are sterile filtered.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F are prepared using CDAP chemistry, and the glycoconjugate of S. pneumoniae serotype 6A is prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F are prepared using CDAP chemistry, and the glycoconjugate of S. pneumoniae serotype 19A is prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F are prepared by CDAP chemistry, and glycoconjugates of S. pneumoniae serotypes 6A and 19A are prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F are prepared by CDAP chemistry, and glycoconjugates of S. pneumoniae serotypes 3, 6A, and 19A are prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 22F, and 23F are prepared by CDAP chemistry, and the glycoconjugate of S. pneumoniae serotype 6A is prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 22F, and 23F are prepared using C DAP chemistry, and glycoconjugates of S. pneumoniae serotype 19A are prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 22F, and 23F are prepared using C DAP chemistry, and glycoconjugates of S. pneumoniae serotypes 6A and 19A are prepared by reductive amination.

In one embodiment of the present invention, glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F, and 23F are prepared by CD AP chemistry, and glycoconjugates of S. pneumoniae serotypes 3, 6A, and 19A are prepared by reductive amination.

In one embodiment, the glycoconjugates of the invention are obtained by eTEC conjugation as described in WO 2014/027302. Said glycoconjugates comprise a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate bond, and wherein the carrier protein is covalently conjugated to the eTEC spacer via an amide bond. The eTEC-linked glycoconjugates of the invention may be represented by the general formula (I):

where the atoms containing the eTEC spacer are contained in the central box.

The eTEC spacer comprises seven linear atoms (i.e., _C (O)NH( _CH2 ) _2SCH2C (O)-) and provides stable thioether and amide linkages between the saccharide and the carrier protein. Synthesis of the eTEC-linked glycoconjugate involves reaction of the activated hydroxyl group of the saccharide with the amino group of a thioalkylamine reagent, such as cystamine or cysteamine or their salts, to form a carbamate linkage with the saccharide to produce a thiolated saccharide. Formation of one or more free sulfhydryl groups occurs by reaction with a reducing agent to produce the activated thiolated saccharide. The reaction of the free sulfhydryl groups of the activated thiolated saccharide with an activated carrier protein containing one or more a-haloacetamide groups on the amino-containing residues results in the formation of a thioether bond to form a conjugate in which the carrier protein is attached to the eTEC spacer via an amide bond.

In the glycoconjugates of the invention, the saccharide may be a polysaccharide or an oligosaccharide. The carrier protein may be selected from any suitable carrier described herein or known to those skilled in the art. In common embodiments, the saccharide is a polysaccharide. In some such embodiments, the carrier protein is CRM _197. In some such embodiments, the glycoconjugate linked to eTEC comprises the capsular polysaccharide of S. pneumoniae serotype 33F.

In particularly preferred embodiments, the eTEC-linked glycoconjugate comprises serotype 33F pneumococcal capsular polysaccharide (Pn33F) that is covalently conjugated to CRM ₁₉₇ via an eTEC spacer (serotype 33F eTEC-linked glycoconjugates).

In some embodiments, the glycoconjugate of S. pneumoniae serotypes 1, 7F, 9V and/or 18C of the invention is O-acetylated. In some embodiments, the glycoconjugate of S. pneumoniae serotypes 1, 7F and 9V is O-acetylated and the glycoconjugate of S. pneumoniae serotype 18C is de-O-acetylated.

In some embodiments, the glycoconjugates of the present invention comprise a saccharide having a molecular weight of from 5 kDa to 2000 kDa. In other such embodiments, the saccharide has a molecular weight of from 50 kDa to 1000 kDa. In other such embodiments, the saccharide has a molecular weight of from 70 kDa to 900 kDa. In other such embodiments, the saccharide has a molecular weight of from 100 kDa to 800 kDa. In other such embodiments, the saccharide has a molecular weight of from 200 kDa to 600 kDa. In other such embodiments, the saccharide has a molecular weight of from 100 kDa to 500 kDa. In other such embodiments, the saccharide has a molecular weight of from 100 kDa to 400 kDa. In other such embodiments, the saccharide has a molecular weight of from 150 kDa to 300 kDa. In additional embodiments, the saccharide has a molecular weight of 5 kDa to 100 kDa; from 10 kDa to 100 kDa; from 20 kDa to 100 kDa; from 30 kDa to 100 kDa; from 40 kDa to 100 kDa; from 50 kDa to 100 kDa; from 60 kDa to 100 kDa; from 70 kDa to 100 kDa; from 80 kDa to 100 kDa; from 90 kDa to 100 kDa; from 5 kDa to 90 kDa; from 5 kDa to 80 kDa; from 5 kDa to 70 kDa; from 5 kDa to 60 kDa; from 5 kDa to 50 kDa; from 5 kDa to 40 kDa; from 5 kDa to 30 kDa; from 5 kDa to 20 kDa or from 5 kDa to 10 kDa. Any integer in any of the above ranges is considered an embodiment of the invention. In some such embodiments, the glycoconjugate is prepared by reductive amination.

In some embodiments, a glycoconjugate of the invention has a molecular weight of 100 kDa to 15,000 kDa. In some embodiments, a glycoconjugate of the invention has a molecular weight of 500 kDa to 10,000 kDa. In some embodiments, a glycoconjugate of the invention has a molecular weight of 2,000 kDa to 10,000 kDa. In some embodiments, a glycoconjugate of the invention has a molecular weight of 3,000 kDa to 8,000 kDa. In some embodiments, a glycoconjugate of the invention has a molecular weight of 3,000 kDa to 5,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of 500 kDa to 10,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of 1,000 kDa to 8,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of 2000 kDa to 8000 kDa or 3000 kDa to 7000 kDa.

The molecular weight of the glycoconjugate is measured by the SEC-MALLS method. Any integer in any of the above ranges is considered as an embodiment of the invention.

Another characteristic of the glycoconjugates of the invention is the amount of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) conjugated to the saccharide, which can be expressed as a range of the amount of conjugated lysine residues (degree of conjugation). Modifications of the carrier protein by lysine due to the formation of covalent bonds between lysine and polysaccharides can be detected by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the starting material of the carrier protein used to prepare the conjugate. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is from 2 to 15. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is from 2 to 13. In one embodiment, the degree of conjugation of the glycoconjugates of the present invention is from 2 to 10. In one embodiment, the degree of conjugation of the glycoconjugates of the present invention is from 2 to 8. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is from 2 to 6. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is from 3 to 10. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is from 3 to 6. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is from 5 to 10. In one embodiment, the degree conjugation degree of the glycoconjugates of the invention is from 8 to 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 4. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 5. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 6. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 10. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is in the range of 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the invention can also be characterized by the ratio (w/w) of the saccharide to the carrier protein. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is from 0.5 to 3. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 0.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 0.9. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 1.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 1.2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 1.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 1.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 2.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 2.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate (w/w) is about 3.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 2.0. In other embodiments, the ratio of saccharide to carrier protein (w/w) is from 0.5 to 1.5. In further embodiments, the ratio of saccharide to carrier protein (w/w) is from 0.8 to 1.2. In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is from 0.9 to 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates and immunogenic compositions of the invention may comprise a free saccharide that is not covalently conjugated to the carrier protein but is nonetheless present in the glycoconjugate composition. The free saccharide may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped by or with it).

In a preferred embodiment, the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 20% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 15% free polysaccharide based on the total amount of polysaccharide.

Glycoconjugates can also be characterized by their molecular weight distribution (K _d ). Size exclusion chromatography (CL-4B) can be used to determine the relative molecular weight distribution of a conjugate. Size exclusion chromatography (SEC) uses gravity fed columns to obtain a molecular weight distribution profile of the conjugates. Large molecules, displaced from the pores into the medium, elute faster than small molecules. Fraction collectors are used to collect the eluate from the column. The fractions are tested colorimetrically using saccharide analysis. To determine K _d , columns are calibrated to establish the fraction in which the molecules are completely displaced (V ₀ ), (K _d =0), and the fraction representing maximum retention (V _i ), (K _d =1). The fraction in which the specified property of the sample (V _e ) is achieved is related to K _d by the formula: K _d = (V _e - V ₀ /(V _i - V ₀ ).

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

Another parameter for characterizing the glycoconjugates of the invention is the frequency of attachment of the sugar chain to the lysine on the carrier protein. For example, in some embodiments, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 4 repeating sugar units of the polysaccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 10 repeating sugar units of the polysaccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 15 repeating sugar units of the polysaccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 25 repeating sugar units of the polysaccharide.

In common embodiments, the carrier protein is CRM ₁₉₇ and at least one covalent bond occurs between the CRM ₁₉₇ and the polysaccharide through the eTEC spacer for every 4, 10, 15, or 25 repeating sugar units of the polysaccharide.

In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 5-10 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 2-7 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 7-12 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 10-15 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 4-8 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 10-20 repeating saccharide units. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 2-25 repeating saccharide units. In frequent embodiments, the carrier protein is CRM ₁₉₇ .

In another embodiment, at least one bond occurs between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeating saccharide units of the polysaccharide. In one embodiment, the carrier protein is CRM _197. Any integer in any of the above ranges is considered an embodiment of the invention.

1.4 Carrier protein according to the invention

One component of the glycoconjugate of the invention is a carrier protein to which the saccharide is conjugated. The terms "carrier protein" or "carrier protein" or "carrier" may be used interchangeably herein. Carrier proteins should be suitable for standard conjugation procedures.

In a preferred embodiment, the glycoconjugate carrier protein is selected from the group consisting of: DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM ₁₉₇ (a non-toxic but antigenically identical variant of diphtheria toxin), the A chain mutant of diphtheria toxin CRM ₁₉₇ (CN1034 95161), other mutants of DT (such as CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218:3838-3844), CRM9, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992); deletions or mutations of Glu-148 to Asp, Gin or Ser and/or Ala 158 to Gly and other mutations disclosed in U.S. Patent Nos. 4,709,017 and 4,950,740; mutations of at least one or more of Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Patent Nos. 5,917,017 and 6,455,673; or a fragment disclosed in U.S. Patent No. 5,843,711 of pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect Immun 63:27 06-2713), including ply detoxified in some manner, such as dPLY-GMBS (WO 2004/081515 and WO 2006/032499) or dPLY-formalin, PhtX including PhtA, PhtB, PhtD, PhtE (the PhtA, PhtB, PhtD or PhtE sequences disclosed in WO 00/37105 and WO 00/39299) and Pht protein fusion products, such as PhtDE fusion products, PhtBE fusion products, Pht A-E (WO 01/98334, WO 03/054007, WO 2009/000826), OMPC (meningococcal outer membrane protein, which is typically extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (from JV. meningitidis), PD (Haemophilus influenzae protein D; see, for example, EP0594610 B) or their immunologically functional equivalents, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816–3824), such as the N19 protein (Baraldoi et al. (2004) Infect Immun 72:4 884-4 887), pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), Clostridium difficile toxin A or B (WO 00/61761), transferrin-binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (in particular its non-toxic mutants (such as glutamic acid-substituted exotoxin A 553) (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)). Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD) may also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g. as described in WO 2004/083251), Escherichia coli LT, E. coli ST and P. aeruginosa exotoxin A. Another suitable carrier protein is Streptococcus C5a peptidase (SCP).

In a preferred embodiment, the glycoconjugate carrier protein is independently selected from the group consisting of TT, DT, DT mutants (such as CRM ₁₉₇ ), PhtX, PhtD, PhtDE fusion products of I. influenzae protein D (in particular those described in WO 01/98334 and WO 03/054007), detoxified pneumolysin, PhtB, N19 protein, PspA, OMPC, C. difficile toxin A or B and PsaA.

In one embodiment, the glycoconjugate carrier protein of the invention is DT (diphtheria toxoid). In another embodiment, the glycoconjugate carrier protein of the invention is TT (tetanus toxoid). In another embodiment, the glycoconjugate carrier protein of the invention is Streptococcus C5a peptidase (SCP). In one embodiment, the glycoconjugate carrier protein of the invention is enzymatically inactive Streptococcus C5a peptidase (SCP).

In another embodiment, the carrier protein of the glycoconjugates of the invention is PD (I. influenzae protein D; see, for example, EP0594610B).

The CRM ₁₉₇ protein is a nontoxic form of diphtheria toxin that is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by Corynebacterium diphtheriae infected with the nontoxic phage β197 ^tox- , created by nitrosoguanidine mutagenesis of the toxigenic corynophage beta (Uchida et al. (1971) Nature New Biology 233:8-11). The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin but differs from it by a single base substitution (guanine to adenine) in the structural gene. This single base substitution results in an amino acid substitution (glutamic acid to glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin. CRM ₁₉₇ protein is a safe and effective T-cell-dependent saccharide carrier.

Further information on CRM ₁₉₇ and its production can be found, for example, in US Patent No. 5,614,382. In one embodiment, the capsular saccharides of the invention are conjugated to a CRM ₁₉₇ protein or an A chain of CRM ₁₉₇ (see CN103495161). In one embodiment, the capsular saccharides of the invention are conjugated to an A chain of CRM ₁₉₇ obtained by expression of genetically recombinant E. coli (see CN103495161). In a preferred embodiment, the capsular saccharides of the invention are conjugated to CRM _197. In one embodiment, all of the capsular saccharides of the invention are conjugated to CRM ₁₉₇ .

Accordingly, in frequent embodiments, the glycoconjugates of the invention comprise CRM ₁₉₇ as a carrier protein, wherein the capsular polysaccharide is covalently linked to CRM ₁₉₇ .

1.5. Glycoconjugates of serotype 15B according to the invention

In one embodiment, the glycoconjugates 15B of the invention are as defined in this section.

Capsular polysaccharides of S. pneumoniae serotype 15B were prepared as described above (see also WO 2015/110942).

In one embodiment, the polysaccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then linked directly or via a spacer (linker) group to an amino group on a carrier protein (preferably CRM ₁₉₇ ). For example, the spacer can be cystamine or cysteamine to form a thiolated polysaccharide that can be linked to the carrier via a thioether linkage obtained by reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyroxy]succinimide (GMBS) ester) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derived saccharide is conjugated to a carrier protein (e.g. CRM ₁₉₇ ) by carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the carrier protein. Such conjugates are described, for example, in WO 93/15760, WO 95/08348 and WO 96/129094.

In one embodiment of the present invention, the S. pneumoniae serotype 15B glycoconjugate is produced using CDAP chemistry.

Other suitable conjugation methods include carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of these are described in International Patent Application Publication No. WO 98/42721. Conjugation may involve a carbonyl linker, which may be formed by reaction of the free hydroxyl group of the saccharide with CDI (see Bethell et al. (1979) 1. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followed by reaction with the protein to form a carbamate linkage. This reaction may involve reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a carbamate intermediate of CDI, and coupling of the carbamate intermediate of CDI to an amino group on the protein.

In a preferred embodiment, the capsular polysaccharide of S. pneumoniae serotype 15B is conjugated to a carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340, and 2007/0184071, WO 2006/110381, WO 2008/079653, WO 2008/143709, WO 2015/110942, and WO 2019/139692). Therefore, in a preferred embodiment, the serotype 15B glycoconjugate of the present invention is produced by reductive amination.

Reductive amination involves two steps as described above.

In some embodiments, the S. pneumoniae serotype 15B glycoconjugate of the present invention comprises a saccharide having a molecular weight of from 5 kDa to 2000 kDa. In other such embodiments, the saccharide has a molecular weight of from 20 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 1000 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 500 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 400 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 300 kDa. In a preferred embodiment, the saccharide has a molecular weight of from 100 kDa to 350 kDa. In another embodiment, the saccharide has a molecular weight of from 100 kDa to 300 kDa. In another embodiment, the saccharide has a molecular weight of from 150 kDa to 300 kDa.

In further embodiments, the saccharide has a molecular weight of from 5 kDa to 100 kDa; from 10 kDa to 100 kDa; from 20 kDa to 100 kDa; from 50 kDa to 100 kDa; or from 90 kDa to 100 kDa. Any integer in any of the above ranges is considered an embodiment of the invention. In some such embodiments, the glycoconjugate is prepared by reductive amination.

In some embodiments, the S. pneumoniae serotype 15B glycoconjugate of the present invention has a molecular weight of 100 kDa to 20,000 kDa. In other such embodiments, the S. pneumoniae serotype 15B glycoconjugate of the present invention has a molecular weight of 200 kDa to 10,000 kDa. In yet other such embodiments, the S. pneumoniae serotype 15B glycoconjugate of the present invention has a molecular weight of 2,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15B glycoconjugate of the invention has a molecular weight of 1,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15B glycoconjugate of the invention has a molecular weight of 1,500 kDa to 4,500 kDa. In further embodiments, the S. pneumoniae serotype 15B glycoconjugate of the invention has a molecular weight of 2000 kDa to 4500 kDa. In further embodiments, the S. pneumoniae serotype 15B glycoconjugate of the invention has a molecular weight of 500 kDa to 2000 kDa.

The molecular weight of the glycoconjugate is measured by the SEC-MALLS method. Any integer in any of the above ranges is considered as an embodiment of the invention.

Another way in which the glycoconjugates of the invention can be characterized is by determining the amount of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) conjugated to the saccharide, which can be expressed as a range of the amount of conjugated lysine residues (degree of conjugation). Modifications of the carrier protein by lysine due to the formation of covalent bonds between lysine and polysaccharides can be detected by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the starting material of the carrier protein used to prepare the conjugate. In a preferred embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 15. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 13. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 10. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 6. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 3 to 10. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 3 to 6. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 5 to 10. In one embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 8 to 12. In a preferred embodiment, the degree of conjugation of the 15B glycoconjugates of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 4. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 5. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 6. In one embodiment, the degree conjugation degree of the glycoconjugate of the invention is about 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 10. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 15. In a preferred embodiment, the degree of conjugation of glycoconjugate 15B of the invention is from 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the invention can also be characterized by the ratio (w/w) of the saccharide to the carrier protein. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is from 0.5 to 3. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is from 0.4 to 2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 0.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 0.9. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 1.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 1.2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 1.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 1.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 2.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 2.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is about 3.0. In other embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15B (w/w) is from 0.5 to 2.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 1.5. In further embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.8 to 1.2. In a preferred embodiment, the ratio of the capsular polysaccharide to the carrier protein in the conjugate is from 0.9 to 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The frequency of attachment of the saccharide chain to the lysine on the carrier protein is another parameter for characterizing the glycoconjugate of S. pneumoniae serotype 15B of the invention. For example, in some embodiments, at least one covalent bond occurs between the carrier protein and the saccharide 15B for every 4 repeating saccharide units of the saccharide 15B. In another embodiment, at least one covalent bond occurs between the carrier protein and the saccharide 15B for every 10 repeating saccharide units of the saccharide 15B. In another embodiment, at least one covalent bond occurs between the carrier protein and the saccharide 15B for every 15 repeating saccharide units of the saccharide 15B. In a further embodiment, at least one covalent bond occurs between the carrier protein and saccharide 15B for every 25 repeating saccharide units of saccharide 15B.

In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 5-10 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 2-7 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 7-12 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 10-15 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 4-8 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 10-20 repeating saccharide units. In other embodiments, the glycoconjugate 15B comprises at least one covalent bond between the carrier protein and the saccharide 15B for every 2-25 repeating saccharide units. In frequent embodiments, the carrier protein is CRM ₁₉₇ .

In another embodiment, at least one bond occurs between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeating saccharide units of the polysaccharide. In one embodiment, the carrier protein is CRM _197. Any integer in any of the above ranges is considered an embodiment of the invention.

In a preferred embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.5 mM acetate per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.6 mM acetate per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.65 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the glycoconjugate comprises at least 0.7 mM acetate per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises 0.5 mM to 0.85 mM acetate per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises 0.6 mM to 0.85 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the serotype 15B glycoconjugate of the invention comprises 0.7 mM to 0.85 mM acetate per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises from 0.8 mM to 0.85 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the presence of O-acetyl groups is determined by ion HPLC analysis.

In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.1 mM glycerol per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.2 mM glycerol per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.5 mM glycerol per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.8 mM glycerol per mM serotype 15B capsular polysaccharide. In one embodiment, the serotype 15B glycoconjugate of the invention comprises at least 0.9 mM glycerol per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the serotype 15B glycoconjugate of the invention comprises 0.6 mM to 1 mM glycerol per mM serotype 15B capsular polysaccharide. In another preferred embodiment, the serotype 15B glycoconjugate of the invention comprises 0.7 mM to 1 mM glycerol per mM serotype 15B capsular polysaccharide. In a most preferred embodiment, the serotype 15B glycoconjugate of the invention comprises 0.8 mM to 1 mM glycerol per mM serotype 15B capsular polysaccharide.

In one embodiment, the serotype 15B glycoconjugate of the invention comprises from 0.5 mM to 0.7 mM glycerol per mM serotype 15B capsular polysaccharide. In another preferred embodiment, the serotype 15B glycoconjugate of the invention comprises about 0.7 mM glycerol per mM serotype 15B capsular polysaccharide.

In a preferred embodiment, the S. pneumoniae serotype 15B glycoconjugate of the invention comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide based on the total amount of polysaccharide. In one embodiment, the 15B glycoconjugate comprises less than about 25% free polysaccharide based on the total amount of polysaccharide. In one embodiment, the 15B glycoconjugate comprises less than about 20% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the 15B glycoconjugate comprises less than about 15% free polysaccharide based on the total amount of polysaccharide.

Glycoconjugates can also be characterized by their molecular weight distribution (K _d ). Size exclusion chromatography (CL-4B) can be used to determine the relative molecular weight distribution of a conjugate. Size exclusion chromatography (SEC) uses gravity fed columns to obtain a molecular weight distribution profile of the conjugates. Large molecules, displaced from the pores into the medium, elute faster than small molecules. Fraction collectors are used to collect the eluate from the column. The fractions are tested colorimetrically using saccharide analysis. To determine K _d , columns are calibrated to establish the fraction in which the molecules are completely displaced (V ₀ ), (K _d =0), and the fraction representing maximum retention (V _i ), (K _d =1). The fraction in which the specified property of the sample (V _e ) is achieved is related to K _d by the formula: K _d = (V _e - V ₀ )/(V _i - V ₀ ).

In a preferred embodiment, at least 30% of the S. pneumoniae serotype 15B glycoconjugate of the invention has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 40% of the 15B glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the 15B glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 60% of the 15B glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, 50% to 80% of the glycoconjugate 15B has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, 65% to 80% of the glycoconjugate 15B has a K _d value that is less than or equal to 0.3 on a CL-4B column.

1.6. Glycoconjugates of serotype 15C according to the invention

In one embodiment, the 15C glycoconjugates of the invention are as defined in this section.

Capsular polysaccharides of S. pneumoniae serotype 15C are prepared as described above (see also WO 2019/139692).

In one embodiment, the polysaccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then linked directly or via a spacer (linker) group to an amino group on a carrier protein (preferably CRM ₁₉₇ ). For example, the spacer can be cystamine or cysteamine to form a thiolated polysaccharide that can be linked to the carrier via a thioether linkage obtained by reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derived saccharide is conjugated to a carrier protein (e.g. CRM ₁₉₇ ) by carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the carrier protein. Such conjugates are described, for example, in WO 93/15760, WO 95/08348 and WO 96/129094.

In one embodiment of the present invention, the S. pneumoniae serotype 15C glycoconjugate is produced using CDAP chemistry.

Other suitable conjugation methods include carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of these are described in International Patent Application Publication No. WO 98/42721. Conjugation may involve a carbonyl linker, which may be formed by reaction of the free hydroxyl group of the saccharide with CDI (see Bethell et al. (1979) 1. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followed by reaction with the protein to form a carbamate linkage. This reaction may involve reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a carbamate intermediate of CDI, and coupling of the carbamate intermediate of CDI to an amino group on the protein.

In a preferred embodiment, the capsular polysaccharide of S. pneumoniae serotype 15C is conjugated to a carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340, and 2007/0184071, WO 2006/110381, WO 2008/079653, WO 2008/143709, WO 2015/110942, and WO 2019/139692). Therefore, in a preferred embodiment, the serotype 15C glycoconjugate of the present invention is produced by reductive amination.

Reductive amination involves two steps as described above.

In some embodiments, the S. pneumoniae serotype 15C glycoconjugate of the present invention comprises a saccharide having a molecular weight of from 5 kDa to 2000 kDa. In other such embodiments, the saccharide has a molecular weight of from 20 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 1000 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 500 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 400 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 300 kDa. In a preferred embodiment, the saccharide has a molecular weight of from 100 kDa to 350 kDa. In another preferred embodiment, the saccharide has a molecular weight of 100 kDa to 300 kDa. In a more preferred embodiment, the saccharide has a molecular weight of 150 kDa to 300 kDa.

In further embodiments, the saccharide has a molecular weight of from 5 kDa to 100 kDa; from 10 kDa to 100 kDa; from 20 kDa to 100 kDa; from 50 kDa to 100 kDa; or from 90 kDa to 100 kDa. Any integer in any of the above ranges is considered an embodiment of the invention. In some such embodiments, the glycoconjugate is prepared by reductive amination.

In some embodiments, the S. pneumoniae serotype 15C glycoconjugate of the present invention has a molecular weight of 100 kDa to 20,000 kDa. In other such embodiments, the S. pneumoniae serotype 15C glycoconjugate of the present invention has a molecular weight of 200 kDa to 10,000 kDa. In yet other such embodiments, the S. pneumoniae serotype 15C glycoconjugate of the present invention has a molecular weight of 2,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15C glycoconjugate of the invention has a molecular weight of 1,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15C glycoconjugate of the invention has a molecular weight of 1,500 kDa to 4,500 kDa. In further embodiments, the S. pneumoniae serotype 15C glycoconjugate of the invention has a molecular weight of 2000 kDa to 4500 kDa. In further embodiments, the S. pneumoniae serotype 15C glycoconjugate of the invention has a molecular weight of 500 kDa to 2000 kDa.

The molecular weight of the glycoconjugate is measured by the SEC-MALLS method. Any integer in any of the above ranges is considered as an embodiment of the invention.

Another way in which the glycoconjugates of the invention can be characterized is by determining the amount of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) conjugated to the saccharide, which can be expressed as a range of the amount of conjugated lysine residues (degree of conjugation). Modifications of the carrier protein by lysine due to the formation of covalent bonds between lysine and polysaccharides can be detected by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the starting material of the carrier protein used to prepare the conjugate. In a preferred embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 15. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 13. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 10. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 6. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 3 to 10. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 3 to 6. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 5 to 10. In one embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 8 to 12. In a preferred embodiment, the degree of conjugation of the 15C glycoconjugates of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 4. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 5. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 6. In one embodiment, the degree conjugation degree of the glycoconjugate of the invention is about 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 10. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 15. In a preferred embodiment, the degree of conjugation of the 15C glycoconjugate of the invention is from 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the invention can also be characterized by the ratio (w/w) of the saccharide to the carrier protein. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is from 0.5 to 3. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is from 0.4 to 2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 0.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 0.9. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 1.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 1.2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 1.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 1.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 2.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 2.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the 15C glycoconjugate (w/w) is about 3.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 2.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 1.5. In further embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.8 to 1.2. In a preferred embodiment, the ratio of the capsular polysaccharide to the carrier protein in the conjugate is from 0.9 to 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The frequency of attachment of the saccharide chain to the lysine on the carrier protein is another parameter for characterizing the glycoconjugate of S. pneumoniae serotype 15C of the invention. For example, in some embodiments, at least one covalent bond occurs between the carrier protein and the 15C saccharide for every 4 repeating saccharide units of the 15C saccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the 15C saccharide for every 10 repeating saccharide units of the 15C saccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the 15C saccharide for every 15 repeating saccharide units of the 15C saccharide. In a further embodiment, at least one covalent bond occurs between the carrier protein and the 15C saccharide for every 25 repeating saccharide units of the 15C saccharide.

In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 5-10 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 2-7 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 7-12 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 10-15 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 4 to 8 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 10 to 20 repeating saccharide units. In other embodiments, the 15C glycoconjugate comprises at least one covalent bond between the carrier protein and the 15C saccharide for every 2 to 25 repeating saccharide units. In frequent embodiments, the carrier protein is CRM ₁₉₇ .

In another embodiment, at least one bond occurs between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeating saccharide units of the polysaccharide. In one embodiment, the carrier protein is CRM _197. Any integer in any of the above ranges is considered an embodiment of the invention.

In one embodiment, the serotype 15C glycoconjugate of the invention comprises at least 0.1 mM glycerol per mM serotype 15C capsular polysaccharide. In one embodiment, the serotype 15C glycoconjugate of the invention comprises at least 0.2 mM glycerol per mM serotype 15C capsular polysaccharide. In one embodiment, the serotype 15C glycoconjugate of the invention comprises at least 0.5 mM glycerol per mM serotype 15C capsular polysaccharide. In one embodiment, the serotype 15C glycoconjugate of the invention comprises at least 0.8 mM glycerol per mM serotype 15C capsular polysaccharide. In one embodiment, the serotype 15C glycoconjugate of the invention comprises at least 0.9 mM glycerol per mM serotype 15C capsular polysaccharide. In a preferred embodiment, the serotype 15C glycoconjugate of the invention comprises 0.6 mM to 1 mM glycerol per mM serotype 15C capsular polysaccharide. In another preferred embodiment, the serotype 15C glycoconjugate of the invention comprises 0.7 mM to 1 mM glycerol per mM serotype 15C capsular polysaccharide. In a most preferred embodiment, the serotype 15C glycoconjugate of the invention comprises 0.8 mM to 1 mM glycerol per mM serotype 15C capsular polysaccharide.

In one embodiment, the serotype 15C glycoconjugate of the invention comprises from 0.5 mM to 0.7 mM glycerol per mM serotype 15C capsular polysaccharide. In another preferred embodiment, the serotype 15C glycoconjugate of the invention comprises about 0.7 mM glycerol per mM serotype 15C capsular polysaccharide.

In a preferred embodiment, the S. pneumoniae serotype 15C glycoconjugate of the invention comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the 15C glycoconjugate comprises less than about 25% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the 15C glycoconjugate comprises less than about 20% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the 15C glycoconjugate comprises less than about 15% free polysaccharide based on the total amount of polysaccharide.

Glycoconjugates can also be characterized by their molecular weight distribution (K _d ). Size exclusion chromatography (CL-4B) can be used to determine the relative molecular weight distribution of a conjugate. Size exclusion chromatography (SEC) uses gravity fed columns to obtain a molecular weight distribution profile of the conjugates. Large molecules, displaced from the pores into the medium, elute faster than small molecules. Fraction collectors are used to collect the eluate from the column. The fractions are tested colorimetrically using saccharide analysis. To determine K _d , columns are calibrated to establish the fraction in which the molecules are completely displaced (V ₀ ), (K _d =0), and the fraction representing maximum retention (V _i ), (K _d =1). The fraction in which the specified property of the sample is achieved (V _e ) is related to K _d by the formula: K _d = (V _e - V ₀ )/(V _i - V ₀ ).

In a preferred embodiment, at least 30% of the S. pneumoniae serotype 15C glycoconjugate of the invention has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, at least 40% of the 15C glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the 15C glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 60% of the 15C glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, 50% to 80% of the 15C glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, 65% to 80% of the 15C glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column.

1.7 Serotype 15A glycoconjugates according to the invention

In one embodiment, the glycoconjugates 15A of the invention are as defined in this section.

Capsular polysaccharides of S. pneumoniae serotype 15A are prepared as described above (see also WO 2019/139692).

In one embodiment, the polysaccharides are activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then linked directly or via a spacer (linker) group to an amino group on a carrier protein (preferably CRM ₁₉₇ ). For example, the spacer can be cystamine or cysteamine to form a thiolated polysaccharide that can be linked to the carrier via a thioether linkage obtained by reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyroxy]succinimide (GMBS) ester) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derived saccharide is conjugated to a carrier protein (e.g. CRM ₁₉₇ ) by carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the carrier protein. Such conjugates are described, for example, in WO 93/15760, WO 95/08348 and WO 96/129094.

In one embodiment of the present invention, the S. pneumoniae serotype 15A glycoconjugate is produced using CDAP chemistry.

Other suitable conjugation methods include carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of these are described in International Patent Application Publication No. WO 98/42721. Conjugation may involve a carbonyl linker, which may be formed by reaction of the free hydroxyl group of the saccharide with CDI (see Bethell et al. (1979) 1. Biol. Chern. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followed by reaction with the protein to form a carbamate linkage. This reaction may involve reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a carbamate intermediate CDI, and coupling of the carbamate intermediate CDI to an amino group on the protein.

In a preferred embodiment, the capsular polysaccharide of S. pneumoniae serotype 15A is conjugated to a carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340, and 2007/0184071, WO 2006/110381, WO 2008/079653, WO 2008/143709, WO 2015/110942, and WO 2019/139692). Therefore, in a preferred embodiment, the serotype 15A glycoconjugate of the present invention is produced by reductive amination.

Reductive amination involves two steps as described above.

In some embodiments, the S. pneumoniae serotype 15A glycoconjugate of the present invention comprises a saccharide having a molecular weight of from 5 kDa to 2000 kDa. In other such embodiments, the saccharide has a molecular weight of from 20 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 1000 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 800 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 500 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 400 kDa. In further such embodiments, the saccharide has a molecular weight of from 100 kDa to 300 kDa. In a preferred embodiment, the saccharide has a molecular weight of from 100 kDa to 350 kDa. In another preferred embodiment, the saccharide has a molecular weight of 100 kDa to 300 kDa. In a more preferred embodiment, the saccharide has a molecular weight of 150 kDa to 300 kDa.

In further embodiments, the saccharide has a molecular weight of from 5 kDa to 100 kDa; from 10 kDa to 100 kDa; from 20 kDa to 100 kDa; from 50 kDa to 100 kDa; or from 90 kDa to 100 kDa. Any integer in any of the above ranges is considered an embodiment of the invention. In some such embodiments, the glycoconjugate is prepared by reductive amination.

In some embodiments, the S. pneumoniae serotype 15A glycoconjugate of the present invention has a molecular weight of 100 kDa to 20,000 kDa. In other such embodiments, the S. pneumoniae serotype 15A glycoconjugate of the present invention has a molecular weight of 200 kDa to 10,000 kDa. In yet other such embodiments, the S. pneumoniae serotype 15A glycoconjugate of the present invention has a molecular weight of 2,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15A glycoconjugate of the invention has a molecular weight of 1,000 kDa to 5,000 kDa. In further embodiments, the S. pneumoniae serotype 15A glycoconjugate of the invention has a molecular weight of 1,500 kDa to 4,500 kDa. In further embodiments, the S. pneumoniae serotype 15A glycoconjugate of the invention has a molecular weight of 2000 kDa to 4500 kDa. In further embodiments, the S. pneumoniae serotype 15A glycoconjugate of the invention has a molecular weight of 500 kDa to 2000 kDa.

The molecular weight of the glycoconjugate is measured by the SEC-MALLS method. Any integer in any of the above ranges is considered as an embodiment of the invention.

Another way in which the glycoconjugates of the invention can be characterized is by determining the amount of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) conjugated to the saccharide, which can be expressed as a range of the amount of conjugated lysine residues (degree of conjugation). Modifications of the carrier protein by lysine due to the formation of covalent bonds between lysine and polysaccharides can be detected by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the starting material of the carrier protein used to prepare the conjugate. In a preferred embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 2 to 15. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 2 to 13. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 2 to 10. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 2 to 6. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 3 to 10. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is from 3 to 6. In one embodiment, the degree of conjugation of the glycoconjugates 15A of the invention is glycoconjugates of the invention is from 5 to 10. In one embodiment, the degree of conjugation of the 15A glycoconjugates of the invention is from 8 to 12. In a preferred embodiment, the degree of conjugation of the 15A glycoconjugates of the invention is from 2 to 8. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 3. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 4. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 5. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 6. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 8. In one embodiment, the degree of conjugation glycoconjugate of the invention is about 10. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 15. In a preferred embodiment, the degree of conjugation of glycoconjugate 15A of the invention is from 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the invention can also be characterized by a ratio (w/w) of saccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in glycoconjugate 15A (w/w) is from 0.5 to 3. In some embodiments, the ratio of polysaccharide to carrier protein in glycoconjugate 15A (w/w) is from 0.4 to 2. In some embodiments, the ratio of polysaccharide to carrier protein in glycoconjugate 15A (w/w) is about 0.8. In some embodiments, the ratio of polysaccharide to carrier protein in glycoconjugate 15A (w/w) is about 0.9. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 1.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 1.2. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 1.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 1.8. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 2.0. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 2.5. In some embodiments, the ratio of the polysaccharide to the carrier protein in the glycoconjugate 15A (w/w) is about 3.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 2.0. In other embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.5 to 1.5. In further embodiments, the ratio of the saccharide to the carrier protein (w/w) is from 0.8 to 1.2. In a preferred embodiment, the ratio of the capsular polysaccharide to the carrier protein in the conjugate is from 0.9 to 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The frequency of attachment of the saccharide chain to the lysine on the carrier protein is another parameter for characterizing the glycoconjugate of S. pneumoniae serotype 15A of the invention. For example, in some embodiments, at least one covalent bond occurs between the carrier protein and saccharide 15A for every 4 repeating saccharide units of saccharide 15A. In another embodiment, at least one covalent bond occurs between the carrier protein and saccharide 15A for every 10 repeating saccharide units of saccharide 15A. In another embodiment, at least one covalent bond occurs between the carrier protein and saccharide 15A for every 15 repeating saccharide units of saccharide 15A. In a further embodiment, at least one covalent bond occurs between the carrier protein and saccharide 15A for every 25 repeating saccharide units of saccharide 15A.

In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 5-10 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 2-7 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 7-12 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 10-15 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 4-8 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 10-20 repeating saccharide units. In other embodiments, the glycoconjugate 15A comprises at least one covalent bond between the carrier protein and the saccharide 15A for every 2-25 repeating saccharide units. In frequent embodiments, the carrier protein is CRM ₁₉₇ .

In another embodiment, at least one bond occurs between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeating saccharide units of the polysaccharide. In one embodiment, the carrier protein is CRM _197. Any integer in any of the above ranges is considered an embodiment of the invention.

In one embodiment, the serotype 15A glycoconjugate of the invention comprises at least 0.1 mM glycerol per mM serotype 15A capsular polysaccharide. In one embodiment, the serotype 15A glycoconjugate of the invention comprises at least 0.2 mM glycerol per mM serotype 15A capsular polysaccharide. In one embodiment, the serotype 15A glycoconjugate of the invention comprises at least 0.5 mM glycerol per mM serotype 15A capsular polysaccharide. In one embodiment, the serotype 15A glycoconjugate of the invention comprises at least 0.8 mM glycerol per mM serotype 15A capsular polysaccharide. In one embodiment, the serotype 15A glycoconjugate of the invention comprises at least 0.9 mM glycerol per mM serotype 15A capsular polysaccharide. In a preferred embodiment, the serotype 15A glycoconjugate of the invention comprises 0.6 mM to 1 mM glycerol per mM serotype 15A capsular polysaccharide. In another preferred embodiment, the serotype 15A glycoconjugate of the invention comprises 0.7 mM to 1 mM glycerol per mM serotype 15A capsular polysaccharide. In a most preferred embodiment, the serotype 15A glycoconjugate of the invention comprises 0.8 mM to 1 mM glycerol per mM serotype 15A capsular polysaccharide.

In one embodiment, the serotype 15A glycoconjugate of the invention comprises from 0.5 mM to 0.7 mM glycerol per mM serotype 15A capsular polysaccharide. In another preferred embodiment, the serotype 15A glycoconjugate of the invention comprises about 0.7 mM glycerol per mM serotype 15A capsular polysaccharide.

In a preferred embodiment, the S. pneumoniae serotype 15A glycoconjugate of the invention comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide based on the total amount of polysaccharide. In one embodiment, the 15A glycoconjugate comprises less than about 25% free polysaccharide based on the total amount of polysaccharide. In one embodiment, the 15A glycoconjugate comprises less than about 20% free polysaccharide based on the total amount of polysaccharide. In a preferred embodiment, the 15A glycoconjugate comprises less than about 15% free polysaccharide based on the total amount of polysaccharide.

Glycoconjugates can also be characterized by their molecular weight distribution (K _d ). Size exclusion chromatography (CL-4B) can be used to determine the relative molecular weight distribution of a conjugate. Size exclusion chromatography (SEC) uses gravity fed columns to obtain a molecular weight distribution profile of the conjugates. Large molecules, displaced from the pores into the medium, elute faster than small molecules. Fraction collectors are used to collect the eluate from the column. The fractions are tested colorimetrically using saccharide analysis. To determine K _d , columns are calibrated to establish the fraction in which the molecules are completely displaced (V ₀ ), (K _d =0), and the fraction representing maximum retention (V _i ), (K _d =1). The fraction in which the specified property of the sample (Ve) is achieved is related to K _d by the formula: K _d = (V _e - V ₀ )/(V _i - V ₀ ).

In one embodiment, at least 30% of the S. pneumoniae serotype 15A glycoconjugate of the invention has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 40% of the 15A glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the 15A glycoconjugate has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, at least 60% of the glycoconjugate 15A has a K _d value that is less than or equal to 0.3 on a CL-4B column. In one embodiment, from 50% to 80% of the glycoconjugate 15A has a K _d value that is less than or equal to 0.3 on a CL-4B column. In a preferred embodiment, from 65% to 80% of the glycoconjugate 15A has a K _d value that is less than or equal to 0.3 on a CL-4B column.

1.8. Combination of glycoconjugates according to the invention

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, a glycoconjugate of S. pneumoniae serotype 15C, and a glycoconjugate of S. pneumoniae serotype 15A.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A. Preferably, said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C when a glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not comprise a capsular saccharide of S. pneumoniae serotype 15B when a glycoconjugate of S. pneumoniae serotype 15C is present.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A. Preferably, said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15C.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A. Preferably, said composition does not comprise a capsular saccharide of S. pneumoniae serotype 15B.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and/or 23F.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of each of the following eight S. pneumoniae serotypes: (1, 4, 6B, 9V, 14, 18C, 19F, and 23F); (4, 5, 6B, 9V, 14, 18C, 19F, and 23F), or (4, 6B, 7F, 9V, 14, 18C, 19F, and 23F).

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following ten S. pneumoniae serotypes: 1, 5, 4, 6B, 7F, 9V, 14, 18C, 19F, and 23F.

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following eleven S. pneumoniae serotypes: (1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, and 23F) or (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F).

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following twelve S. pneumoniae serotypes: 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following thirteen S. pneumoniae serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype 22F.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype 33F.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype 8.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype 10A.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype HA.

In one embodiment, any of the above immunogenic compositions further comprises at least one glycoconjugate of S. pneumoniae serotype 12F.

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following two serotypes of S. pneumoniae:

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following three serotypes of S. pneumoniae:

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following four serotypes of S. pneumoniae:

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following five S. pneumoniae serotypes:

In one embodiment, any of the above immunogenic compositions comprises at least one glycoconjugate of each of the following six S. pneumoniae serotypes: 22F and 33F, and 12F, and 10A, and 11A, and 8.

In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates of S. pneumoniae serotype 2.

In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates of S. pneumoniae serotype 17F.

In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates of S. pneumoniae serotype 20.

Preferably, all glycoconjugates of the above immunogenic composition are individually conjugated to a carrier protein.

In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15B glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15C glycoconjugate is conjugated to CRM _{197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15A glycoconjugate is conjugated to CRM 197. In} one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 22F glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 33F glycoconjugate is conjugated to CRM ₁₉₇ . In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 12F glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 10A glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype HA glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 8 glycoconjugate is conjugated to CRM ₁₉₇ . In one embodiment, any of the above immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 6A and 19A are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the glycoconjugates of S. pneumoniae serotype 3 are conjugated to CRM ₁₉₇ . In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 2 glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 17F glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 20 glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15C glycoconjugates are conjugated to CRM ₁₉₇ .

In one embodiment, all of the glycoconjugates of the above immunogenic compositions are individually conjugated to CRM ₁₉₇ .

In one embodiment, the S. pneumoniae serotype 18C glycoconjugate of any of the above immunogenic compositions is individually conjugated to TT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogenic compositions are individually conjugated to PD.

In one embodiment, the S. pneumoniae serotype 19F glycoconjugate of any of the above immunogenic compositions is conjugated to DT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogenic compositions are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT.

In one embodiment, said immunogenic composition comprises 2 to 25 different serotypes of S. pneumoniae. In one embodiment, said immunogenic composition comprises glycoconjugates of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 different pneumococcal serotypes.

In one embodiment, said immunogenic composition comprises glycoconjugates of 2 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 3 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 4 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 5 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 6 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 7 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 8 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 9 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 10 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 11 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 12 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 13 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 14 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 15 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 16 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 17 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 18 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 19 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 20 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 21 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 22 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 23 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 24 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 25 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 16 or 20 different serotypes.

In one embodiment, said immunogenic composition is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 20-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 21-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 22-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 2"-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 23-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 24-valent pneumococcal conjugate composition.

Preferably, all glycoconjugates of the immunogenic composition of the invention are individually conjugated to a carrier protein. In one embodiment, the glycoconjugates of the immunogenic composition of the invention are individually conjugated to CRM _197. In one embodiment, all glycoconjugates of the immunogenic composition of the invention are individually conjugated to CRM ₁₉₇ .

1.9. Set of immunogenic compositions according to the invention

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15B, wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15C is as defined in Section 1.6. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In another preferred embodiment, said S. pneumoniae serotype 15C corresponds to the definition given in section 1.6 and said S. pneumoniae serotype 15A corresponds to the definition given in section 1.7.

In another embodiment, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15C, wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15B is as defined in Section 1.5. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In another preferred embodiment, said S. pneumoniae serotype 15B corresponds to the definition given in section 1.5 and said S. pneumoniae serotype 15A corresponds to the definition given in section 1.7.

In another embodiment, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15B is as defined in Section 1.5. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In a preferred embodiment, said S. pneumoniae serotype 15C is as defined in Section 1.6. In an even more preferred embodiment, said S. pneumoniae serotype 15B corresponds to the definition given in section 1.5, said S. pneumoniae serotype 15C corresponds to the definition given in section 1.6, and said S. pneumoniae serotype 15A corresponds to the definition given in section 1.7.

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15B, wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15C is as defined in Section 1.6. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In another preferred embodiment, said S. pneumoniae serotype 15C is as defined in section 1.6 and said S. pneumoniae serotype 15A is as defined in section 1.7. In one embodiment, said S. pneumoniae serotype 15C glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15A glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15A and 15C glycoconjugates are conjugated to CRM ₁₉₇ .

In another embodiment, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not comprise a capsular saccharide of S. pneumoniae serotype 15C, wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15B is as defined in Section 1.5. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In another preferred embodiment, said S. pneumoniae serotype 15B is as defined in section 1.5 and said S. pneumoniae serotype 15A is as defined in section 1.7. In one embodiment, said S. pneumoniae serotype 15B glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15A glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15A and 15B glycoconjugates are conjugated to CRM ₁₉₇ .

In one aspect, the invention relates to a kit of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration. In a preferred embodiment, said immunogenic compositions are intended for parallel, concomitant or sequential administration. In a preferred embodiment, said S. pneumoniae serotype 15B is as defined in Section 1.5. In a preferred embodiment, said S. pneumoniae serotype 15C is as defined in Section 1.6. In a preferred embodiment, said S. pneumoniae serotype 15A is as defined in Section 1.7. In an even more preferred embodiment, said S. pneumoniae serotype 15B meets the definition set out in section 1.5, said S. pneumoniae serotype 15C meets the definition set out in section 1.6, and said S. pneumoniae serotype 15A meets the definition set out in section 1.7. In one embodiment, said S. pneumoniae serotype 15B glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15C glycoconjugate is conjugated to CRM _197. In one embodiment, said S. pneumoniae serotype 15A glycoconjugate is conjugated to CRM ₁₉₇ . In one embodiment, said glycoconjugates of S. pneumoniae serotypes 15A, 15B and 15C are conjugated to CRM ₁₉₇ .

By "simultaneous administration" is meant the administration of therapeutically effective doses of the first and second immunogenic compositions in a single unit dosage form.

By "concurrent administration" is meant the administration of therapeutically effective doses of the first and second immunogenic compositions through the same access site, but in separate unit dosage forms within a short period of time of each other. Concurrent administration essentially represents the administration of two immunogenic compositions at approximately the same time, but in separate unit dosage forms, through the same access site. Concurrent administration of the first and second immunogenic compositions is often performed during the same physician visit.

By "concomitant administration" is meant the administration of therapeutically effective doses of the first and second immunogenic compositions in separate unit dosage forms at a short interval from each other to different anatomical sites. Concomitant administration essentially represents the administration of two immunogenic compositions at approximately the same time, but in separate unit dosage forms and to different anatomical sites. Concomitant administration of the first and second immunogenic compositions is often performed during the same physician visit.

By "sequential administration" is meant administration of a therapeutically effective dose of only the first or only the second immunogenic composition, followed by administration of a therapeutically effective dose of the remaining immunogenic composition after a time interval of at least about 1 month. For example, in one embodiment, the first immunogenic composition is administered as a single dosage form, and then, at an interval of at least about 1 month, the second immunogenic composition is administered as a separate single dosage form. In an alternative embodiment, the second immunogenic composition is administered as a single dosage form, and then, at an interval of at least about 1 month, the first immunogenic composition is administered as a separate single dosage form. Sequential administration of the first and second immunogenic compositions is often performed during separate physician visits.

1. In one embodiment, said first immunogenic composition further comprises, in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

2. In another embodiment, said first immunogenic composition further comprises, to paragraph 1 above, glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F.

3. In another embodiment, said first immunogenic composition further comprises, in addition to points 1 or 2 above, glycoconjugates of S. pneumoniae serotypes 6A and 19A.

4. In another embodiment, said first immunogenic composition further comprises, in addition to items 1, 2 or 3 above, a glycoconjugate of S. pneumoniae serotype 3.

5. In another embodiment, said first immunogenic composition further comprises, in addition to items 1, 2, 3 or 4 above, a glycoconjugate of S. pneumoniae serotype 22F.

6. In another embodiment, said first immunogenic composition further comprises, in addition to items 1, 2, 3, 4 or 5 above, a glycoconjugate of S. pneumoniae serotype 33F.

In one embodiment, said first immunogenic composition, in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, further comprises glycoconjugates of S. pneumoniae serotypes 8, 10A, 11A, 12F, 22F and 33F.

Preferably, all glycoconjugates of the above first immunogenic compositions are individually conjugated to a carrier protein.

In one embodiment, any of the above first immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 6A and 19A are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the glycoconjugates of S. pneumoniae serotype 3 are conjugated to CRM ₁₉₇ . In one embodiment, any of the above first immunogenic compositions, the S. pneumoniae serotype 22F glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the S. pneumoniae serotype 33F glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the S. pneumoniae serotype 10A glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the S. pneumoniae serotype HA glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above first immunogenic compositions, the S. pneumoniae serotype 12F glycoconjugates are conjugated to CRM ₁₉₇ . In one embodiment of any of the above first immunogenic compositions, the S. pneumoniae serotype 8 glycoconjugates are conjugated to CRM ₁₉₇ .

In one embodiment, all of the glycoconjugates of any of the above first immunogenic compositions are individually conjugated to CRM ₁₉₇ .

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above first immunogenic compositions are individually conjugated to PD.

In one embodiment, the S. pneumoniae serotype 18C glycoconjugate of any of the above first immunogenic compositions is conjugated to TT.

In one embodiment, the S. pneumoniae serotype 19F glycoconjugate of any of the above first immunogenic compositions is conjugated to DT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above first immunogenic compositions are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above first immunogenic compositions are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT, the glycoconjugate of S. pneumoniae serotype 22F is conjugated to CRM ₁₉₇ and the glycoconjugate of S. pneumoniae serotype 33F is conjugated to CRM ₁₉₇ .

In one embodiment, the first immunogenic composition comprises 7 to 25 different serotypes of S. pneumoniae. In one embodiment, the first immunogenic composition comprises 7 to 20 different serotypes of S. pneumoniae. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 7 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 8 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 10 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 15 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 16 different serotypes. In one embodiment, the first immunogenic compositions comprise glycoconjugates of 20 different serotypes. In one embodiment, the first immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 7-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is an 8-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 9-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 10-valent pneumococcal conjugate composition. In one embodiment, the aforementioned first immunogenic composition is an 11-valent pneumococcal conjugate composition. In one embodiment, the aforementioned first immunogenic composition is a 12-valent pneumococcal conjugate composition. In one embodiment, the aforementioned first immunogenic composition is a 13-valent pneumococcal conjugate composition. In one embodiment, the aforementioned first immunogenic composition is a 14-valent pneumococcal conjugate composition. In one embodiment, the aforementioned first immunogenic composition is a 15-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 17-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is an 18-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 20-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 21-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 22-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 23-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 24-valent pneumococcal conjugate composition. In one embodiment, the first immunogenic composition is a 25-valent pneumococcal conjugate composition.

In one embodiment, the first immunogenic composition is an 8-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said first immunogenic composition is a 14-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said first immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said first immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, the above-mentioned first immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, the said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said first immunogenic composition is a 20-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, the first immunogenic compositions further comprise one or more adjuvants as described below in Section 4.

In one embodiment, the above-mentioned first immunogenic compositions are prepared as described below in Section 5.

1. In one embodiment, said second immunogenic composition, in addition to the glycoconjugate of S. pneumoniae serotype 15A, further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

2. In another embodiment, said second immunogenic composition further comprises, in addition to paragraph 1 above, glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F.

3. In another embodiment, said second immunogenic composition comprises, in addition to points 1 or 2 above, glycoconjugates of S. pneumoniae serotypes 6A and 19A.

4. In another embodiment, said second immunogenic composition further comprises, in addition to items 1, 2 or 3 above, a glycoconjugate of S. pneumoniae serotype 3.

5. In another embodiment, said second immunogenic composition further comprises, in addition to items 1, 2, 3 or 4 above, a glycoconjugate of S. pneumoniae serotype 22F.

6. In another embodiment, said second immunogenic composition further comprises, in addition to items 1, 2, 3, 4 or 5 above, a glycoconjugate of S. pneumoniae serotype 33F.

Preferably, all glycoconjugates of the above second immunogenic compositions are individually conjugated to a carrier protein.

In one embodiment, any of the above second immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the glycoconjugates of S. pneumoniae serotypes 6A and 19A are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the glycoconjugates of S. pneumoniae serotype 3 are conjugated to CRM ₁₉₇ . In one embodiment, any of the above second immunogenic compositions, the S. pneumoniae serotype 22F glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the S. pneumoniae serotype 33F glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the S. pneumoniae serotype 10A glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the S. pneumoniae serotype 11A glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above second immunogenic compositions, the S. pneumoniae serotype 12F glycoconjugates are conjugated to CRM ₁₉₇ . In one embodiment of any of the above second immunogenic compositions, the S. pneumoniae serotype 8 glycoconjugates are conjugated to CRM ₁₉₇ .

In one embodiment, all of the glycoconjugates of any of the above second immunogenic compositions are individually conjugated to CRM ₁₉₇ .

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogenic compositions are individually conjugated to PD.

In one embodiment, the S. pneumoniae serotype 18C glycoconjugate of any of the above second immunogenic compositions is conjugated to TT.

In one embodiment, the S. pneumoniae serotype 19F glycoconjugate of any of the above second immunogenic compositions is conjugated to DT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogenic compositions are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT.

In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above second immunogenic compositions are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT, the glycoconjugate of S. pneumoniae serotype 22F is conjugated to CRM ₁₉₇ and the glycoconjugate of S. pneumoniae serotype 33F is conjugated to CRM ₁₉₇ .

In one embodiment, the second immunogenic composition comprises 7 to 25 different serotypes of S. pneumoniae. In one embodiment, the second immunogenic composition comprises 7 to 20 different serotypes of S. pneumoniae. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 7 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 8 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 10 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 15 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 16 different serotypes. In one embodiment, the second immunogenic compositions comprise glycoconjugates of 20 different serotypes. In one embodiment, the second immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 7-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is an 8-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 9-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 10-valent pneumococcal conjugate composition. In one embodiment, the aforementioned second immunogenic composition is an 11-valent pneumococcal conjugate composition. In one embodiment, the aforementioned second immunogenic composition is a 12-valent pneumococcal conjugate composition. In one embodiment, the aforementioned second immunogenic composition is a 13-valent pneumococcal conjugate composition. In one embodiment, the aforementioned second immunogenic composition is a 14-valent pneumococcal conjugate composition. In one embodiment, the above-mentioned second immunogenic composition is a 15-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 17-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is an 18-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 20-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 21-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 22-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 23-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 24-valent pneumococcal conjugate composition. In one embodiment, the second immunogenic composition is a 25-valent pneumococcal conjugate composition.

In one embodiment, the second immunogenic composition is an 8-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 14-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 20-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, the second immunogenic composition is a 9-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated to CRM ₁₉₇ .

In one embodiment, the above-mentioned second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F individually conjugated to CRM ₁₉₇ .

In one embodiment, the above-mentioned second immunogenic composition is a 17-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, said second immunogenic composition is a 21-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition also comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

In one embodiment, the second immunogenic compositions further comprise one or more adjuvants as described below in Section 4.

In one embodiment, the above second immunogenic compositions are prepared as described below in Section 5.

1.10 Additional vaccine for achieving optimal protection against S. pneumoniae serotypes 15A, 15B and 15C according to the invention

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 15B and 15C.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 15A and 15C.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 15A and 15B.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise a glycoconjugate of S. pneumoniae serotype 15B.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise a glycoconjugate of S. pneumoniae serotype 15C.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise a glycoconjugate of S. pneumoniae serotype 15A.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15B, 15C, 18C, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 15C, 18C, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 15B, 18C, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15B, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15C, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15B, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15C, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 15B, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 15C, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 15A, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, IF, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, IF, 8, 9V, 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15A, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

Preferably, all glycoconjugates of the above immunogenic composition are individually conjugated to a carrier protein.

In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15C glycoconjugate is conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15B glycoconjugates are conjugated to CRM _197. In one embodiment, any of the above immunogenic compositions, the S. pneumoniae serotype 15A glycoconjugates are conjugated to CRM ₁₉₇ .

In one embodiment, the above immunogenic composition comprises glycoconjugates of 2 to 25 different serotypes of S. pneumoniae. In one embodiment, the above immunogenic composition comprises glycoconjugates of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different pneumococcal serotypes.

In one embodiment, said immunogenic composition comprises glycoconjugates of 2 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 3 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 4 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 5 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 6 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 7 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 8 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 9 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 10 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 11 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 12 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 13 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 14 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 15 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 16 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 17 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 18 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 19 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 20 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 21 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 22 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 23 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 24 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 25 different pneumococcal serotypes. In one embodiment, said immunogenic composition comprises glycoconjugates of 16 or 20 different serotypes.

In one embodiment, said immunogenic composition is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 20-valent pneumococcal conjugate composition. In one embodiment, said immunogenic composition is a 21-valent pneumococcal conjugate composition.

All glycoconjugates of the immunogenic composition of the invention are preferably individually conjugated to a carrier protein. In one embodiment, the glycoconjugates of the immunogenic composition of the invention are individually conjugated to CRM _197. In one embodiment, all glycoconjugates of the immunogenic composition of the invention are individually conjugated to CRM ₁₉₇ .

The immunogenic compositions provided in this section are also particularly useful as an adjunct to prior art vaccines to achieve optimal protection against S. pneumoniae serotypes 15A, 15B, and 15C.

In particular, the immunogenic compositions provided in this section are particularly useful for complementing prior art vaccines such as Prevnar®, Prevnarl3® or Synflorix® to achieve optimal protection against S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 10-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, and 23F are conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 12-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 13-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic compositions of this section are intended for use as an adjunct to a 14-valent pneumococcal conjugate vaccine, wherein said conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 15-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 19-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions of this section are intended for use as an adjunct to a vaccine that comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 20-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 22-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 23-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B are conjugated to CRM ₁₉₇ .

In one embodiment, the immunogenic compositions provided in this section are intended for use as an adjunct to a 24-valent pneumococcal conjugate vaccine, wherein the conjugates comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B.

In one embodiment, said glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B are conjugated to CRM ₁₉₇ .

By "add-on" or "add-on" to a specific vaccine is meant the use of an immunogenic composition to extend the spectrum of protection provided by said vaccine against serotype(s) of S. pneumoniae against which said vaccine does not provide significant protection. In particular, the immunogenic compositions provided in this section can be used as an adjunct to a vaccine to achieve significant protection against S. pneumoniae serotypes 15A, 15B and/or 15C, preferably against S. pneumoniae serotypes 15A, 15B and 15C.

Thus, in one embodiment, the immunogenic compositions provided in this section that comprise glycoconjugates of S. pneumoniae serotypes 15B and 15C are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15A but does not comprise glycoconjugates of S. pneumoniae serotypes 15B and 15C.

In one embodiment, the immunogenic compositions provided in this section that comprise glycoconjugates of S. pneumoniae serotypes 15A and 15C are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15B but does not comprise glycoconjugates of S. pneumoniae serotypes 15A and 15C.

In one embodiment, the immunogenic compositions provided in this section that comprise glycoconjugates of S. pneumoniae serotypes 15A and 15B are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15C but does not comprise glycoconjugates of S. pneumoniae serotypes 15A and 15B.

In one embodiment, the immunogenic compositions provided in this section that comprise a glycoconjugate of S. pneumoniae serotype 15C are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not comprise a glycoconjugate of S. pneumoniae serotypes 15C.

In one embodiment, the immunogenic compositions provided in this section that comprise a glycoconjugate of S. pneumoniae serotype 15C are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15A but does not comprise a glycoconjugate of S. pneumoniae serotype 15C.

In one embodiment, the immunogenic compositions provided in this section that comprise a glycoconjugate of S. pneumoniae serotype 15C are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15B but does not comprise a glycoconjugate of S. pneumoniae serotype 15C.

In one embodiment, the immunogenic compositions provided in this section comprising a glycoconjugate of S. pneumoniae serotype 15B are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise a glycoconjugate of S. pneumoniae serotypes 15B.

In one embodiment, the immunogenic compositions provided in this section that comprise a glycoconjugate of S. pneumoniae serotype 15B are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15A but does not comprise a glycoconjugate of S. pneumoniae serotype 15B.

In one embodiment, the immunogenic compositions provided in this section comprising a glycoconjugate of S. pneumoniae serotype 15B are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15C but does not comprise a glycoconjugate of S. pneumoniae serotype 15B.

In one embodiment, the immunogenic compositions provided in this section comprising a glycoconjugate of S. pneumoniae serotype 15A are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not comprise a glycoconjugate of S. pneumoniae serotypes 15A.

In one embodiment, the immunogenic compositions provided in this section comprising a glycoconjugate of S. pneumoniae serotype 15A are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15B but does not comprise a glycoconjugate of S. pneumoniae serotype 15A.

In one embodiment, the immunogenic compositions provided in this section comprising a glycoconjugate of S. pneumoniae serotype 15A are intended for use as an adjunct to a pneumococcal conjugate vaccine that comprises a glycoconjugate of S. pneumoniae serotype 15C but does not comprise a glycoconjugate of S. pneumoniae serotype 15A.

2. Dosage of immunogenic compositions

2.1. Amount of polysaccharides

The amount of glycoconjugate(s) in each dose is selected such that the amount will elicit an immunoprotective response without causing significant adverse side effects in typical vaccinated subjects. This amount will vary depending on the specific immunogen used and how it is presented.

The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on the total polysaccharide for that conjugate (conjugated and unconjugated). For example, a glycoconjugate with 20% free polysaccharide will contain approximately 80 μg conjugated polysaccharide and approximately 20 μg unconjugated polysaccharide in a 100 μg dose. The amount of glycoconjugate may vary depending on the serotype of the streptococcus. The concentration of saccharide can be determined using a uronic acid assay.

The "immunogenic amount" of the different polysaccharide components in the immunogenic composition may vary and may independently be about 1.0 μg, about 2.0 μg, about 3.0 μg, about 4.0 μg, about 5.0 μg, about 6.0 μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, about 10.0 μg, about 15.0 μg, about 20.0 μg, about 30.0 μg, about 40.0 μg, about 50.0 μg, about 60.0 μg, about 70.0 μg, about 80.0 μg, about 90.0 μg, or about 100.0 μg of any particular polysaccharide antigen.

Typically, each dose may contain from 0.1 μg to 100 μg of the polysaccharide of the serotype, in particular from 0.5 μg to 20 μg, more particularly from 1 μg to 10 μg, and even more particularly from 2 μg to 5 μg. Any integer in any of the above ranges is considered as an embodiment of the invention.

In one embodiment, each dose may contain 1 mcg, 2 mcg, 3 mcg, 4 mcg, 5 mcg, 6 mcg, 7 mcg, 8 mcg, 9 mcg, 10 mcg, 15 mcg, or 20 mcg of the polysaccharide of the serotype.

2.2. Amount of media

Typically, each dose may contain from 5 μg to 150 μg of carrier protein, in particular from 10 μg to 100 μg of carrier protein, more particularly from 15 μg to 100 μg of carrier protein, more particularly from 25 to 75 μg of carrier protein, more particularly from 30 μg to 70 μg of carrier protein, more particularly from 30 to 60 μg of carrier protein, more particularly from 30 μg to 50 μg of carrier protein and even more particularly from 40 to 60 μg of carrier protein. In one embodiment, said carrier protein is CRM ₁₉₇ .

In one embodiment, each dose may contain about 25 mcg, about 26 mcg, about 27 mcg, about 28 mcg, about 29 mcg, about 30 mcg, about 31 mcg, about 32 mcg, about 33 mcg, about 34 mcg, about 35 mcg, about 36 mcg, about 37 mcg, about 38 mcg, about 39 mcg, about 40 mcg, about 41 mcg, about 42 mcg, about 43 mcg, about 44 mcg, about 45 mcg, about 46 mcg, about 47 mcg, about 48 mcg, about 49 mcg, about 50 mcg, about 51 mcg, about 52 mcg, about 53 mcg, about 54 mcg, about 55 mcg, about 56 mcg, about 57 mcg, about 58 mcg, about 59 mcg, about 60 mcg, about 61 mcg, about 62 mcg, about 63 mcg, about 64 mcg, about 65 mcg, about 66 mcg, about 67 mcg, 68 mcg, about 69 mcg, about 70 mcg, about 71 mcg, about 72 mcg, about 73 mcg, about 74 mcg, or about 75 mcg of carrier protein. In one embodiment, said carrier protein is CRM ₁₉₇ .

3. Other antigens

The immunogenic compositions of the invention comprise conjugated saccharide antigens of S. pneumoniae (glycoconjugates). They may also include antigens of other pathogens, in particular bacteria and/or viruses. Preferred additional antigens are selected from: diphtheria toxoid (D), tetanus toxoid (T), pertussis antigen (P), which is usually acellular (Pa), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis A virus (HAV) antigen, conjugated capsular saccharide of Haemophilus influenzae type b (Hib), inactivated poliovirus vaccine (IPV).

In one embodiment, the immunogenic compositions of the invention comprise D-T-Pa. In one embodiment, the immunogenic compositions of the invention comprise D-T-Pa-Hib, D-T-Pa-IPV, or D-T-Pa-HBsAg. In one embodiment, the immunogenic compositions of the invention comprise D-T-Pa-HBsAg-IPV or D-T-Pa-HBsAg-Hib. In one embodiment, the immunogenic compositions of the invention comprise D-T-Pa-HBsAg-IPV-Hib.

4. Adjuvant(s)

In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant (e.g., one, two, or three adjuvants). The term "adjuvant" refers to a compound or mixture that enhances an immune response to an antigen. Antigens may act primarily as a delivery system, primarily as an immunomodulator, or may exhibit both potent properties. Suitable adjuvants include adjuvants suitable for use in mammals, including humans.

Examples of known suitable adjuvants of the delivery system type 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(0, L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

In one embodiment, the immunogenic compositions disclosed herein comprise aluminum salts (alum) (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide) as an adjuvant. In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In one embodiment, the immunogenic compositions disclosed herein comprise 0.1 mg/mL to 1 mg/mL or 0.2 mg/mL to 0.3 mg/mL of elemental aluminum in the form of aluminum phosphate. In one embodiment, the immunogenic compositions disclosed herein comprise about 0.25 mg/mL of elemental aluminum in the form of aluminum phosphate.

Examples of known suitable immunomodulatory type adjuvants that can be used in humans include, but are not limited to, saponin extracts from the bark of the Aquilla tree (QS21, Quil A), TLR4 agonists such as MPL (monophosphoryl lipid A), 3DMPL (3-O-deacylated MPL) or GLA-AQ, LT/CT mutants, cytokines such as various interleukins (e.g., IL-2, IL-12) or GM-CSF, etc.

Examples of known suitable immunomodulatory-type adjuvants having both delivery and immunomodulatory properties that can be used in humans include, but are not limited to, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 2006/134423, and WO 2007/026190), or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications involving, but not limited to, animal experiments, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), emulsigen, N-acetylmuramyl-G-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-G-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton, can be used. (MPL+TDM+CWS) in 2% squalene/Tween 80 emulsion.

Other illustrative adjuvants for enhancing the efficacy of the pneumococcal vaccines disclosed herein include, but are not limited to: (1) oil-in-water emulsion formulations (with other specific immunostimulatory agents such as muramyl dipeptides (see below) or bacterial cell wall components), such as, for example, (a) SAF containing 10% squalane, 0.4% Tween 80, 5% Pluronic block polymer L121, and thr-MDP, either microfluidized to form a submicron emulsion or vortexed to form a larger particle size emulsion, and (b) RIBI™ Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more components bacterial cell wall such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), Abisco® (Isconova, Sweden) or Iscomatrix® (Commonwealth Serum Laboratories, Australia) or particles derived therefrom such as ISCOMs (immunostimulatory complexes) may be used, wherein the ISCOMs may be free of additional detergent (e.g. WO 00/07621); (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636)), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see e.g. GB-2220221, EP0689454), optionally substantially free of alum when used with pneumococcal saccharides (see e.g. WO 00/56358); (6) combinations of 3dMPL, for example with QS21 and/or oil-in-water emulsions (see for example EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or polyoxyethylene ester (see for example WO 99/52549); (8) a polyoxyethylene ester surfactant in combination with octoxynol (WO 01/21207) or a polyoxyethylene ether or alkyl ether surfactant in combination with at least one further non-ionic surfactant such as octoxynol (WO 01/21152); (9) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO 00/62800); (10) an immunostimulant and a metal salt particle (see, for example, WO 00/23105); (11) a saponin and an oil-in-water emulsion, for example, WO 99/11241; (12) a saponin (for example, QS21)+3dMPL+IM2 (optionally+sterol), for example, WO 98/57 659; (13) other substances that act as immunostimulatory agents to enhance the effectiveness of the composition. Muramyl peptides include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxy phosphoryloxy)ethylamine MTP-PE), etc.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a CpG oligonucleotide as an adjuvant. A CpG oligonucleotide as used herein refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and accordingly, these terms are used interchangeably unless otherwise indicated. Immunostimulatory CpG oligodeoxynucleotides comprise one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally in certain preferred base contexts. The methylation status of an immunostimulatory CpG motif typically refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide comprising at least one unmethylated CpG dinucleotide is an oligonucleotide that comprises a 5'-unmethylated cytosine linked by a phosphate bond to a 3'-guanine and that activates the immune system through binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotide may comprise one or more methylated CpG dinucleotides, which will activate the immune system through TLR9, but not as strongly as if the CpG motif(s) were/were unmethylated. The immunostimulatory CpG oligonucleotides may comprise one or more palindromes, which in turn may include a CpG dinucleotide. CpG oligonucleotides are described in a number of 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.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise any of the CpG oligonucleotides described in WO 2010/125480 on page 3, line 22 - page 12, line 36.

Various classes of CpG immunostimulatory oligonucleotides have been identified. These classes are designated as classes A, B, C and P and are described in more detail in WO 2010/125480 at page 3, line 22 to page 12, line 36. The methods of the invention encompass the use of these various classes of CpG immunostimulatory oligonucleotides.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class A CpG oligonucleotide. In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class B CpG oligonucleotide.

The class B CpG oligonucleotide sequences of the invention are described in detail above and are also disclosed in published WO 96/02555, WO 98/18810, and U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. Exemplary sequences include, but are not limited to, those sequences disclosed in these referenced applications and patents.

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

In any of these sequences, all of the linkages may be phosphorothioate linkages. In another embodiment, in any of these sequences, one or more linkages may be phosphodiester linkages, preferably between the "C" and "G" of a CpG motif, forming a semi-soft CpG oligonucleotide. In any of these sequences, the 5'-T may be substituted with ethyluridine or a halogen; examples of halogen substitution include, but are not limited to, bromouridine or iodouridine substitutions.

Some non-limiting examples of class B oligonucleotides include:

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class C CpG oligonucleotide.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise a class P CpG oligonucleotide.

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

In one embodiment, all internucleotide linkages of the CpG oligonucleotides disclosed herein are phosphodiester linkages ("soft" oligonucleotides as described in WO 2007/026190). In another embodiment, the CpG oligonucleotides of the invention are rendered resistant to degradation (e.g., stabilized). A "stabilized oligonucleotide" refers to an oligonucleotide that is relatively resistant to degradation in vivo (e.g., by an exo- or endonuclease). Nucleic acid stabilization can be achieved by backbone modifications. An oligonucleotide with phosphorothioate linkages has maximal activity, wherein the oligonucleotide is protected from degradation by intracellular exo- and endonucleases.

Immunostimulatory oligonucleotides may have a chimeric backbone that has combinations of phosphodiester and phosphorothioate linkages. For the purposes of the present invention, a chimeric backbone refers to a partially stabilized backbone in which at least one internucleotide linkage is a phosphodiester linkage or a linkage similar to a phosphodiester linkage, and where at least one other internucleotide linkage is a stabilized internucleotide linkage, where at least one phosphodiester linkage or a linkage similar to a phosphodiester linkage and at least one stabilized linkage are different. When the phosphodiester linkage is preferably located within a CpG motif, such molecules are called "semi-soft" as described in WO 2007/026190.

Other modified oligonucleotides include combinations of phosphodiester, phosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate and/or para-ethoxy linkages.

The mixed backbone ODN can be synthesized as described in WO 2007/026190.

The size of the CpG oligonucleotide (i.e., the number of nucleotide residues along the length of the oligonucleotide) can also contribute to the stimulatory activity of the oligonucleotide. To facilitate cellular uptake, the CpG oligonucleotides of the invention preferably have a minimum length of 6 nucleotide residues. Oligonucleotides of any size greater than 6 nucleotides (even those several kb in length) are capable of inducing an immune response if they contain a sufficient number of immunostimulatory motifs, since longer oligonucleotides are degraded within cells. In some embodiments, the CpG oligonucleotides are 6 to 100 nucleotides in length, preferably 8 to 30 nucleotides. In important embodiments, the nucleic acids and oligonucleotides of the invention are not plasmids or expression vectors.

In one embodiment, the CpG oligonucleotide disclosed herein comprises substitutions or modifications, for example, in bases and/or sugars, as described in WO 2007/026190 at paragraphs 134-147.

In one embodiment, a CpG oligonucleotide of the present invention is chemically modified. Examples of chemical modifications are known in the art and are described, for example, in Uhlmann et al. (1990) Chem. Rev. 90:543; S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and Hunziker et al., (1995) Mod. Synth. Methods 7:331-417. The oligonucleotide of the invention may have one or more modifications, wherein each modification is localized in a specific phosphodiester internucleoside bridge and/or in a specific [3-D-ribose unit and/or in a specific position of the natural nucleoside base compared to an oligonucleotide of the same sequence that consists of natural DNA or RNA.

In some embodiments of the invention, CpG-containing nucleic acids may be simply mixed with immunogenic carriers according to methods known to those skilled in the art (see, for example, WO 03/024480).

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

5. Composition

The immunogenic compositions of the invention may be prepared in liquid form (i.e., in the form of solutions or suspensions) or in lyophilized form. Liquid formulations can advantageously be administered directly from the form in which they are packaged and are therefore ideally suited for injection without the need for reconstitution in an aqueous medium, as is required for lyophilized compositions of the invention.

The immunogenic composition of the present invention can be formulated by methods known in the art. For example, individual pneumococcal conjugates can be formulated in a physiologically acceptable carrier to form the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

The present disclosure provides an immunogenic composition comprising any combination of glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

In one embodiment, the immunogenic composition of the invention is in liquid form, preferably in aqueous liquid form.

The immunogenic compositions of the present invention may comprise one or more of a buffer, a salt, a divalent cation, a nonionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or chelating agent, or any combination thereof.

In one embodiment, the immunogenic composition of the invention comprises a buffer. In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate, succinate, histidine, or citrate buffer. In some embodiments, the buffer is succinate with a final concentration of 1 mM to 10 mM. In one particular embodiment, the final concentration of the succinate buffer is about 5 mM.

In one embodiment, the immunogenic composition of the invention comprises a salt. In some embodiments, the salt is selected from the group consisting of magnesium chloride, potassium chloride, sodium chloride, and a combination thereof. In one particular embodiment, the salt is sodium chloride. In one particular embodiment, the immunogenic composition of the invention comprises 150 mM sodium chloride.

In one embodiment, the immunogenic compositions of the invention comprise a surfactant. In one embodiment, the surfactant is selected from the group consisting of polysorbate 20 (TWEEN™20), polysorbate 40 (TWEEN™40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65), polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-1 01, TRITON™ X-100, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate condensate, polyoxyethylene 660-hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene 35-ricinoleate (CREMOPHOR® EL), soy lecithin, and poloxamer. In one particular embodiment, the surfactant is polysorbate 80. In some of these embodiments, the final concentration of polysorbate 80 in the composition is in the range of at least 0.0001% to 10% polysorbate 80 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 80 in the composition is in the range of 0.001% to 1% polysorbate 80 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 80 in the composition is in the range of 0.01% to 1% polysorbate 80 by weight (w/w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.02% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.01% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.03% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 0.04% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the composition is 0.05% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the composition is 1% polysorbate 80 (w/w).

In one particular embodiment, the surfactant is polysorbate 20. In some of these embodiments, the final concentration of polysorbate 20 in the composition is in the range of 0.0001% to 10% polysorbate 20 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 20 in the composition is in the range of 0.001% to 1% polysorbate 20 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 20 in the composition is in the range of 0.01% to 1% polysorbate 20 by weight (w/w). In other embodiments, the final concentration of polysorbate 20 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 20 (w/w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.02% polysorbate 20 (w/w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.01% polysorbate 20 (w/w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.03% polysorbate 20 (w/w). In another embodiment, the final concentration of polysorbate 20 in the formulation is 0.04% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 20 in the composition is 0.05% polysorbate 20 (w/w). In another embodiment, the final concentration of polysorbate 20 in the composition is 1% polysorbate 20 (w/w).

In one particular embodiment, the surfactant is polysorbate 40. In some of these embodiments, the final concentration of polysorbate 40 in the composition is in the range of 0.0001% to 10% polysorbate 40 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 40 in the composition is in the range of 0.001% to 1% polysorbate 40 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 40 in the composition is in the range of 0.01% to 1% polysorbate 40 by weight (w/w). In other embodiments, the final concentration of polysorbate 40 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 40 (w/w). In another embodiment, the final concentration of polysorbate 40 in the formulation is 1% polysorbate 40 (w/w).

In one particular embodiment, the surfactant is polysorbate 60. In some of these embodiments, the final concentration of polysorbate 60 in the composition is in the range of 0.0001% to 10% polysorbate 60 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 60 in the composition is in the range of 0.001% to 1% polysorbate 60 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 60 in the composition is in the range of 0.01% to 1% polysorbate 60 by weight (w/w). In other embodiments, the final concentration of polysorbate 60 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 60 (w/w). In another embodiment, the final concentration of polysorbate 60 in the formulation is 1% polysorbate 60 (w/w).

In one particular embodiment, the surfactant is polysorbate 65. In some of these embodiments, the final concentration of polysorbate 65 in the formulation is in the range of 0.0001% to 10% polysorbate 65 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 65 in the formulation is in the range of 0.001% to 1% polysorbate 65 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 65 in the formulation is in the range of 0.01% to 1% polysorbate 65 by weight (w/w). In other embodiments, the final concentration of polysorbate 65 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 65 (w/w). In another embodiment, the final concentration of polysorbate 65 in the formulation is 1% polysorbate 65 (w/w).

In one particular embodiment, the surfactant is polysorbate 85. In some of these embodiments, the final concentration of polysorbate 85 in the composition is in the range of 0.0001% to 10% polysorbate 85 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 85 in the composition is in the range of 0.001% to 1% polysorbate 85 by weight (w/w). In some of these embodiments, the final concentration of polysorbate 85 in the composition is in the range of 0.01% to 1% polysorbate 85 by weight (w/w). In other embodiments, the final concentration of polysorbate 85 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 85 (w/w). In another embodiment, the final concentration of polysorbate 85 in the formulation is 1% polysorbate 85 (w/w).

In some embodiments, the immunogenic composition of the invention has a pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even more preferably a pH of 5.8 to 6.0.

In one embodiment of the present invention, a container filled with any of the immunogenic compositions described herein is provided. In one embodiment, the container is selected from the group consisting of a vial, a syringe, a flask, a fermenter, a bioreactor, a bag, a jar, an ampoule, a cartridge, and a disposable pen. In some embodiments, the container is siliconized.

In one embodiment, the container of the present invention is made of glass, metals (e.g., steel, stainless steel, aluminum, etc.) and/or polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In one embodiment, the container of the present invention is made of glass.

In one embodiment of the present invention, a syringe filled with any of the immunogenic compositions described herein is provided. In some embodiments, the syringe is siliconized and/or made of glass.

A typical dose of the immunogenic composition of the invention for injection has a volume of from 0.1 ml to 2 ml, more preferably from 0.2 ml to 1 ml, even more preferably a volume of about 0.5 ml.

Thus, the container or syringe as defined above is filled with a volume of from 0.1 ml to 2 ml, more preferably from 0.2 ml to 1 ml, even more preferably a volume of about 0.5 ml of any of the immunogenic compositions provided herein.

6. The ability of the immunogenic compositions or set of immunogenic compositions according to the invention to induce the production of cross-reactive antibodies

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in humans that are capable of binding to the polysaccharide of S. pneumoniae serotypes 15A, 15B and 15C, as determined by an ELISA assay.

In the ELISA (enzyme-linked immunosorbent assay) method, antibodies from the serum of vaccinated subjects are incubated with polysaccharides adsorbed on a solid support. Bound antibodies are detected using secondary antibodies conjugated to an enzyme used for detection.

In one embodiment, said ELISA assay is a standardized ELISA assay as defined in the WHO document "Training Manual For Enzyme Linked Immunosorbent Assay For The Quantitation Of Streptococcus Pneumoniae Serotype Specific IgG (Pn PS ELISA)" (available at https://www.vaccine.uab.edu/uploads/mdocs/ELISAProtocol(007sp).pdf, accessed September 16, 2020).

ELISA allows quantitative determination of type-specific IgG antibodies to the capsular polysaccharide (PS) of S. pneumoniae present in human serum. When dilutions of human serum are added to microtiter plates coated with type-specific capsular PS, antibodies specific for this capsular PS bind to the microtiter plates. Antibodies bound to the plates are detected using goat anti-human IgG labeled with alkaline phosphatase, followed by the addition of the substrate p-nitrophenyl phosphate. The optical density of the stained final product is proportional to the amount of antibodies to capsular PS present in the serum.

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15A at a concentration of at least 0.05 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.3 μg/mL, 0.35 μg/mL, 0.4 μg/mL, or 0.5 μg/mL, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15A at a concentration of at least 0.3 μg/mL, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15A at a concentration of at least 0.35 μg/ml, as determined by an ELISA assay.

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15B at a concentration of at least 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15B at a concentration of at least 0.3 μg/ml, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15B at a concentration of at least 0.35 μg/ml, as determined by an ELISA assay.

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15C at a concentration of at least 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15C at a concentration of at least 0.3 μg/ml, as determined by an ELISA assay. In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing IgG antibodies in a human that are capable of binding to the polysaccharide of S. pneumoniae serotype 15C at a concentration of at least 0.35 μg/ml, as determined by an ELISA assay.

In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of inducing functional antibodies in humans that are capable of killing S. pneumoniae serotypes 15A, 15B, and 15C, as determined by an in vitro opsonophagocytic assay (OPA) (see Example 1). In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of inducing functional antibodies in humans that are capable of killing S. pneumoniae serotype 15A, as determined by an in vitro opsonophagocytic assay (OPA). In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of inducing functional antibodies in humans that are capable of killing S. pneumoniae serotype 15B, as determined by an in vitro opsonophagocytic assay (OPA). In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of inducing functional antibodies in humans that are capable of killing S. pneumoniae serotype 15C, as determined by an in vitro opsonophagocytic assay (OPA).

The pneumococcal opsonophagocytic response (OPA) assay, which quantifies the killing of S. pneumoniae cells by phagocytic effector cells in the presence of functional antibodies and complement, is considered an important indicator for assessing the efficacy of pneumococcal vaccines.

An in vitro opsonophagocytic assay (OPA) can be performed by coincubating a mixture of Streptococcus pneumoniae cells, heat-inactivated human serum to be tested, differentiated HL-60 cells (phagocytes), and an exogenous source of complement (e.g., baby rabbit complement). Opsonophagocytosis occurs during incubation, and bacterial cells coated with antibody and complement are killed by opsonophagocytosis. Colony-forming units (CFU) of surviving bacteria that escape opsonophagocytosis are determined by plating the assay mixture. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial counts compared with control wells without test serum. The OPA titer is interpolated from the two dilutions that span this 50% killing threshold.

In this type of OPA killing assay, a positive result is defined as an endpoint titer of 1:8 or greater.

In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B, and 15C in at least 50% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B, and 15C in at least 60% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA).

In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B, and 15C in at least 80% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, an immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B, and 15C in at least 90% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B and 15C in at least 95% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of eliciting an antibody titer of at least 1:8 to S. pneumoniae serotypes 15A, 15B and 15C in at least 98% of subjects, as determined by an in vitro opsonophagocytic killing assay (OPA).

In some embodiments, subjects may have OPA serotype-specific antibody titers prior to pneumococcal vaccination, such as due to natural exposure to S. pneumoniae (e.g., in the case of adult subjects).

Thus, it is possible to compare the OPA activity of serum before and after immunization using the immunogenic composition of the invention and compare their responses to serotypes 15A, 15B and 15C to assess the potential increase in the number of responders (see Example 1).

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention significantly increases the proportion of responders (i.e., individuals with serum with an antibody titer of at least 1:8, as determined by in vitro OPA) compared to a pre-immunized population.

Thus, in one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of significantly increasing the proportion of responders to S. pneumoniae serotypes 15A, 15B and 15C (i.e., individuals with a serum titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunized population.

Comparison of the OPA activity of serum before and after immunization using the immunogenic composition of the invention can also be performed by comparing the potential increase in OPA titers.

Thus, it is possible to compare the OPA activity of serum before and after immunization using the immunogenic composition or set of immunogenic compositions of the invention and compare their response to serotypes 15A, 15B and 15C in order to evaluate the effectiveness of increasing OPA titers (see Example 1).

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of significantly increasing the OPA titer in humans compared to a pre-immunized population.

Therefore, in one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of significantly increasing OPA titers against S. pneumoniae serotypes 15A, 15B and 15C in humans compared to a pre-immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5 or 8.0. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 2.0. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 3.0. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 4.0. In one embodiment, the fold increase in the titer of OPA against S. pneumoniae serotype 15B is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, or 8.0. In one embodiment, the fold increase in the titer of OPA against S. pneumoniae serotype 15B is at least 2.0. In one embodiment, the fold increase in the titer of OPA against S. pneumoniae serotype 15B is at least 3.0. In one embodiment, the fold increase in the titer of OPA against S. pneumoniae serotype 15B is at least 4.0. In one embodiment, the fold increase in the titer of OPA against S. pneumoniae serotype 15C is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, or 8.0. In one embodiment, the fold increase in the titer of the OPA against S. pneumoniae serotype 15C is at least 2.0. In one embodiment, the fold increase in the titer of the OPA against S. pneumoniae serotype 15C is at least 3.0. In one embodiment, the fold increase in the titer of the OPA against S. pneumoniae serotype 15C is at least 4.0.

In one embodiment, the immunogenic composition or set of immunogenic compositions of the invention is capable of significantly increasing OPA titers in humans against S. pneumoniae serotypes 15A, 15B and 15C compared to a pre-immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, or 8.0, the fold increase in OPA titer against S. pneumoniae serotype 15B is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, or 8.0, and the fold increase in OPA titer against S. pneumoniae serotype 15C is at least 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, or 8.0. In one embodiment, the fold increase in the OPA titer against S. pneumoniae serotype 15A is at least 2.0, the fold increase in the OPA titer against S. pneumoniae serotype 15B is at least 2.0, and the fold increase in the OPA titer against S. pneumoniae serotype 15C is at least 2.0. In one embodiment, the fold increase in the OPA titer against S. pneumoniae serotype 15A is at least 3.0, the fold increase in the OPA titer against S. pneumoniae serotype 15B is at least 3.0, and the fold increase in the OPA titer against S. pneumoniae serotype 15C is at least 3.0. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 15A is at least 4.0, the fold increase in OPA titer against S. pneumoniae serotype 15B is at least 4.0, and the fold increase in OPA titer against S. pneumoniae serotype 15C is at least 4.0.

7. Use of immunogenic compositions and a set of immunogenic compositions according to the invention

In one embodiment, the immunogenic compositions or set of immunogenic compositions disclosed herein are intended for use as a medicine.

In particular, the immunogenic compositions or set of immunogenic compositions disclosed herein can be used to prevent, treat, or alleviate in a subject an infection, disease, or condition caused by S. pneumoniae.

Thus, in one aspect, the invention relates to a method for preventing, treating or ameliorating in a subject an infection, disease or condition associated with S. pneumoniae, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention relates to a method for preventing, treating or ameliorating in a subject an infection, disease or condition associated with S. pneumoniae, comprising administering to the subject an immunologically effective amount of a set of immunogenic compositions of the invention.

In one aspect, the invention relates to a method for preventing, treating or ameliorating in a subject an infection, disease or condition associated with S. pneumoniae serotypes 15A, 15B and 15C, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention relates to a method for preventing, treating or alleviating in a subject an infection, disease or condition associated with S. pneumoniae serotypes 15A, 15B and 15C, comprising administering to the subject an immunologically effective amount of a set of immunogenic compositions of the invention.

In one aspect, the invention relates to a method for inducing an immune response in a subject to S. pneumoniae serotypes 15A, 15B and 15C, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention relates to a method for inducing an immune response in a subject to S. pneumoniae serotypes 15A, 15B and 15C, comprising administering to the subject an immunologically effective amount of a set of immunogenic compositions of the invention.

In one aspect, the immunogenic compositions of the present invention are for use in a method for preventing, treating or ameliorating in a subject an infection, disease or condition caused by S. pneumoniae serotypes 15A, 15B and 15C.

In one aspect, the immunogenic composition kits of the present invention are for use in a method for preventing, treating or ameliorating in a subject an infection, disease or condition caused by S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, any of the immunogenic compositions disclosed herein is for use in a method of immunizing a subject against S. pneumoniae serotypes 15A, 15B, and 15C infection.

In one embodiment, any set of immunogenic compositions disclosed herein is for use in a method of immunizing a subject against S. pneumoniae serotypes 15A, 15B, and 15C infection.

In one aspect, the present invention is directed to the use of the immunogenic composition disclosed herein for the preparation of a medicament for the prevention, treatment or amelioration in a subject of an infection, disease or condition caused by S. pneumoniae serotypes 15A, 15B and 15C.

In one aspect, the present invention is directed to the use of the immunogenic composition kit disclosed herein for the preparation of a medicament for the prevention, treatment or amelioration in a subject of an infection, disease or condition caused by S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the present invention is directed to the use of the immunogenic composition disclosed herein for the preparation of a medicament for immunizing a subject against S. pneumoniae serotypes 15A, 15B and 15C infection.

In one embodiment, the present invention is directed to the use of the immunogenic compositions disclosed herein for the preparation of a medicament for immunizing a subject against S. pneumoniae serotypes 15A, 15B and 15C infection.

In one aspect, the present invention relates to a method for inducing an immune response to S. pneumoniae serotypes 15A, 15B, and 15C in a subject. In one embodiment, said method comprises administering to the subject an immunogenic composition disclosed herein. In another embodiment, said method comprises administering to the subject a set of immunogenic compositions disclosed herein.

In one embodiment, the immunogenic compositions disclosed herein are vaccines. In one embodiment, the set of immunogenic compositions disclosed herein is a set of vaccines (i.e., the immunogenic compositions of the set of immunogenic compositions are vaccines).

In one embodiment, the immunogenic compositions disclosed herein are for use as a vaccine. In one embodiment, a set of immunogenic compositions disclosed herein is for use as a vaccine. More particularly, the immunogenic compositions or sets of immunogenic compositions disclosed herein can be used to prevent S. pneumoniae serotype 15A, 15B, and 15C infections in a subject. Thus, in one aspect, the invention provides a method of preventing S. pneumoniae serotype 15A, 15B, and 15C infection in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition or set of immunogenic compositions of the invention. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In some such embodiments, the infection is pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, or brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, a cat, a sheep, a pig, a horse, cattle, or a dog. Preferably, said subject is a human.

In one aspect, the immunogenic compositions or kits of immunogenic compositions disclosed herein are for use in a method of preventing, treating or ameliorating in a subject an infection, disease or condition associated with S. pneumoniae serotypes 15A, 15B and 15C. 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, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess. In some such embodiments, the infection, disease or condition is pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, 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, a cat, a sheep, a pig, a horse, a cattle or a dog. Preferably, said subject is a human.

In one aspect, the immunogenic compositions or kits of immunogenic compositions disclosed herein are for use in a method of preventing an infection caused by S. pneumoniae serotypes 15A, 15B and 15C in a subject. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, a cat, a sheep, a pig, a horse, a cattle or a dog. Preferably, said subject is a human.

In one aspect, the present invention is directed to the use of an immunogenic composition or a set of immunogenic compositions disclosed herein for the manufacture of a medicament for the prevention, treatment or amelioration of an infection, disease or condition associated with S. pneumoniae serotypes 15A, 15B and 15C in a subject. Preferably, said subject is a human. 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, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess. In some such embodiments, the infection, disease, or condition is pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, or brain abscess.

In one aspect, the present invention is directed to the use of an immunogenic composition or a set of immunogenic compositions disclosed herein for the preparation of a medicament for preventing an infection caused by S. pneumoniae serotypes 15A, 15B and 15C in a subject. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, a cat, a sheep, a pig, a horse, a cattle or a dog. Preferably, said subject is a human.

The immunogenic compositions or kits of immunogenic compositions of the present invention can be used to protect or treat a human susceptible to infection with S. pneumoniae serotypes 15A, 15B, and 15C by systemic or mucosal administration of the immunogenic compositions or kits of immunogenic compositions. In one embodiment, the immunogenic compositions or kits of immunogenic compositions disclosed herein are administered intramuscularly, intraperitoneally, intradermally, or subcutaneously. In one embodiment, the immunogenic compositions or kits of immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In one embodiment, the immunogenic compositions or kits of immunogenic compositions disclosed herein are administered by intramuscular or subcutaneous injection. In one embodiment, the immunogenic compositions or sets of immunogenic compositions disclosed herein are administered by intramuscular injection. In one embodiment, the immunogenic compositions or sets of immunogenic compositions disclosed herein are administered by subcutaneous injection.

8. A subject to be treated with immunogenic compositions or sets of immunogenic compositions of the invention

As disclosed herein, the immunogenic compositions or sets of immunogenic compositions disclosed herein can be used in various therapeutic or prophylactic methods for the prevention, treatment, or amelioration of a bacterial infection, disease, or condition in a subject.

In a preferred embodiment, said subject is a human. In a most preferred embodiment, said subject is a newborn (i.e., under three months of age), an infant (i.e., between three months and one year of age), or a toddler (i.e., between one year and four years of age).

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

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

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

In one embodiment, the subject to be vaccinated is an immunocompromised individual, in particular a human. An immunocompromised individual is generally understood to be a human who exhibits a weakened or reduced ability to maintain normal humoral or cellular defence against infectious agents.

In one embodiment of the present invention, the subject to be vaccinated is immunocompromised and suffers from a disease or condition that weakens the immune system and results in the production of antibodies that are insufficient to protect against or treat pneumococcal diseases.

In one embodiment, said disease is a primary immunodeficiency disorder. Preferably, said primary immunodeficiency disorder is selected from the group consisting of: combined T- and B-cell immunodeficiencies, antibody deficiencies, well-defined syndromes, diseases due to immune dysregulation, phagocytic disorders, congenital immunodeficiencies, autoinflammatory diseases, and complement deficiencies. In one embodiment, said primary immunodeficiency disorder is a combined T- and B-cell immunodeficiency, antibody deficiency, well-defined syndromes, diseases due to immune dysregulation, phagocytic disorders, congenital immunodeficiencies, autoinflammatory diseases, or complement deficiencies. In one embodiment, said primary immunodeficiency disorder is selected from the disorders described in WO 2010/125480 on page 24, line 11 - page 25, line 19.

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

In one embodiment of the present invention, the subject to be vaccinated is immunocompromised and suffers from nutritional deficiency.

In a particular embodiment of the present invention, the immunocompromised subject to vaccination receives a drug or treatment that reduces the body's resistance to infection. In one embodiment, said drug is selected from the drugs described in WO 2010/125480 on page 26, line 33 - page 26, line 4.

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

In a specific embodiment of the present invention, the white blood cell count (leukocyte count) of the immunocompromised subject to be vaccinated is below 5×10 ⁹ cells per liter, or below 4×10 ⁹ cells per liter, or below 3×10 ⁹ cells per liter, or below 2×10 ⁹ cells per liter, or below 1×10 ⁹ cells per liter, or below 0.5×109 cells per liter, or below 0.3×10 ⁹ cells per liter, or below 0.1×10 ⁹ cells per liter.

White blood cell count (WBC): The number of white blood cells (WBC) in your blood. WBC is usually measured as part of a complete blood count. White blood cells are the blood cells that fight infection in the blood and are different from the red (oxygen-carrying) blood cells known as RBCs. There are different types of white blood cells, including neutrophils (polymorphonuclear leukocytes; PMN), band cells (slightly immature neutrophils), T-cell lymphocytes (T cells), B-cell lymphocytes (B cells), monocytes, eosinophils, and basophils. The white blood cell count reflects all of these cell types. The normal range for white blood cell counts is usually between 4,300 and 10,800 cells per cubic millimeter of blood. It may also be referred to as the white blood cell count and can be expressed in international units as 4.3 x 10, 8 x 10 ⁹ cells per liter.

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

Low white blood cell count, or “neutropenia,” is a condition characterized by abnormally low levels of neutrophils in the circulating blood. Neutrophils are a special type of white blood cell that helps prevent and fight infections. The most common cause of neutropenia in cancer patients is a side effect of chemotherapy. Chemotherapy-induced neutropenia increases a patient’s risk of infection and interferes with cancer treatment.

In a specific embodiment of the present invention, the subject to be vaccinated has a weakened immune system and a CD4+ cell count below 500/ ^mm3 , or a CD4+ cell count below 300/ ^mm3 , or a CD4+ cell count below 200/ ^mm3 , a CD4+ cell count below 100/ ^mm3 , a CD4+ cell count below 75/ ^mm3 , or a CD4+ cell count below 50/ ^mm3 .

CD4 cell count tests usually report the number of cells in mm3. The normal CD4 count is between 500 and 1600, and the CD8 count is between 375 and 1100. In people with HIV, the CD4 count drops dramatically.

In one embodiment, any of the immunocompromised subjects mentioned herein is a human male or a human female. In one embodiment, any of the immunocompromised subjects mentioned herein is a human male. In one embodiment, any of the immunocompromised subjects mentioned herein is a human female.

9. Mode of administration of the immunogenic composition according to the invention

In some cases, only one dose of the immunogenic composition of the invention is required, but in some circumstances, such as severe immunodeficiency states, a second, third or fourth dose may be administered. After the initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

In one embodiment, the vaccination schedule with the immunogenic composition of the invention is a single dose administration. In a particular embodiment, said single dose administration schedule is for healthy individuals at least 2 years of age.

In one embodiment, the vaccination schedule with the immunogenic composition of the invention is a multiple dose schedule. In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 2 months. In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 1 month, or a series of 2 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of from about 1 month to about 2 months. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month, or a series of 3 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of from about 1 month to about 2 months, followed by a fourth dose administered about 10-13 months after the first dose. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month, followed by a fourth dose administered about 10-13 months after the first dose, or a series of 3 doses separated by an interval of about 2 months, followed by a fourth dose administered about 10-13 months after the first dose.

In one embodiment, the multiple dose schedule includes administering at least one dose (e.g., 1, 2, or 3 doses) in the first year of life, followed by at least one dose when the child reaches toddlerhood.

In one embodiment, said multiple dose schedule consists of a series of 2 or 3 doses separated by an interval of about 1 month to about 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 12-18 months of age. In one embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 to 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 2 months of age and then at 12-15 months of age. In another embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 2 months, beginning at 2 months of age, followed by a dose at 12-18 months of age.

In one embodiment, the multiple dose schedule consists of a 4-dose vaccine series at 2, 4, 6, and 12-15 months of age.

In one embodiment, the primary dose is administered on day 0 and one or more booster doses are administered at intervals of about 2 to about 24 weeks, preferably at a dosing interval of 4-8 weeks.

In one embodiment, a primary dose is administered on day 0 and a booster dose is administered approximately 3 months later.

10. Schedule of immunization with a set of immunogenic compositions according to the invention.

In some cases, only one dose of the immunogenic compositions of the invention is required, but in some circumstances, such as severe immunodeficiency or immune immaturity, a second, third or fourth dose may be administered. After the initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

In one embodiment, the vaccination schedule with the set of immunogenic compositions of the invention is a single dose administration. In a particular embodiment, said single dose administration schedule is for healthy individuals at least 2 years of age.

In one embodiment, the vaccination schedule with the set of immunogenic compositions of the invention is a multiple-dose schedule. A multiple-dose schedule is often used in conditions such as immunodeficiency (e.g., in the elderly or immunocompromised) or immune immaturity (e.g., in neonates (i.e., under three months of age), infants (i.e., 3 months to 1 year of age), or toddlers (i.e., 1 year to 4 years of age)). In a particular embodiment, said multiple-dose schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 12 months. In a particular embodiment, said multiple-dose schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said multiple-dose schedule consists of a series of 2 doses separated by an interval of about 1 month. In a particular embodiment, said multiple-dose schedule consists of a series of 2 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 12 months.

In a specific embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 6 months.

In another embodiment, said multiple dose schedule consists of a series of 4 doses, wherein each dose is separated by an interval of from about 1 month to about 12 months.

In a specific embodiment, said multiple dose schedule consists of a series of 4 doses, wherein each dose is separated by an interval of from about 1 month to about 6 months.

In another embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 4 months, followed by administration of a fourth dose approximately 10 to 13 months after the first dose.

In another embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months, followed by administration of a fourth dose approximately 10 to 13 months after the first dose.

In one embodiment, the multiple dose schedule includes administering at least one dose (e.g., 1, 2, or 3 doses) in the first year of life, followed by administering at least one dose when the child reaches toddlerhood.

In one embodiment, the multiple dose schedule consists of a series of 2 or 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 12-18 months of age. In one embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 2 months of age and further at 12-15 months of age. In another embodiment, said multiple dose schedule consists of a series of 2 doses, separated by an interval of about 2 months, beginning at 2 months of age, followed by a dose at 12-18 months of age.

In one embodiment, the multiple dose schedule consists of a 4-dose vaccine series at 2, 4, 6, and 12-15 months of age.

In one embodiment, the primary dose is administered on day 0 and one or more booster doses are administered at dose intervals of about 2 to about 24 weeks, preferably at a dosing interval of 4-8 weeks.

In one embodiment, a primary dose is administered on day 0 and a booster dose is administered approximately 3 months later.

In another embodiment, said multiple dose schedule consists of a series of 5 doses, wherein each dose is separated by an interval of from about 1 month to about 12 months.

In a specific embodiment, said multiple dose schedule consists of a series of 5 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a specific embodiment, said multiple dose schedule consists of a series of 5 doses, where each dose is separated by an interval of about 1, 2, 3, 4, 5 or 6 months. In a specific embodiment, said multiple dose schedule consists of a series of 5 doses, where each dose is separated by an interval of about 1 month, or a series of 5 doses, where each dose is separated by an interval of about 2 months.

In one aspect, the invention relates to any set of immunogenic compositions of the invention for simultaneous, parallel, concomitant or sequential administration with a second immunogenic composition.

In one aspect of the present invention, the first immunogenic composition of the set of immunogenic compositions of the invention is administered in a simultaneous, parallel, concomitant or sequential administration regimen with the second immunogenic composition.

In one aspect of the present invention, the first immunogenic composition of the set of immunogenic compositions of the invention is administered simultaneously with the second immunogenic composition.

In one aspect of the present invention, the first immunogenic composition of the set of immunogenic compositions of the invention is administered in parallel with the second immunogenic composition.

In one aspect of the present invention, the first immunogenic composition of the set of immunogenic compositions of the invention is administered in a concomitant administration regimen with a second immunogenic composition.

In one aspect of the present invention, a first immunogenic composition from a set of immunogenic compositions of the invention is administered sequentially with a second immunogenic composition.

In some cases, only one dose of each of the immunogenic compositions is required, but in some circumstances, a second, third, or fourth dose of one or each of the immunogenic compositions may be administered. Following the initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

In one embodiment, the present invention relates to a set of immunogenic compositions of the invention for use in concomitant administration of a first and second immunogenic composition.

In one embodiment, the vaccination schedule for concomitant administration is a single dose administration (the administration of the first and second immunogenic compositions, although in separate unit dosage forms, is considered a single dose for the purposes of determining the immunization schedule). In a particular embodiment, said single dose administration schedule is for healthy individuals at least 2 years of age.

In one embodiment, the concomitant administration schedule of vaccination is a multiple dose schedule (the administration of the first and second immunogenic compositions, although in separate unit dosage forms, is considered a single dose for the purposes of determining the immunization schedule). In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 12 months. In a particular embodiment, said schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said schedule consists of a series of 2 doses separated by an interval of about 1 month. In a particular embodiment, said schedule consists of a series of 2 doses separated by an interval of about 2 months.

In another embodiment, said schedule of administration of multiple doses consists of a series of 3 doses, where each dose is separated by an interval of from about 1 month to about 12 months. In a particular embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In another embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of about 1 month. In another embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of about 2 months.

In a particular embodiment, said multiple dose schedule consists of a series of 4 doses separated by an interval of from about 1 month to about 12 months. In a particular embodiment, said multiple dose schedule consists of a series of 4 doses separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said multiple dose schedule consists of a series of 4 doses separated by an interval of about 1 month. In a particular embodiment, said multiple dose schedule consists of a series of 4 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 4 months, followed by administration of a fourth dose about 10-13 months after administration of the first dose. In another embodiment, said schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months, followed by administration of a fourth dose about 10-13 months after administration of the first dose. In another embodiment, said schedule consists of a series of 3 doses, wherein each dose is separated by an interval of about 1 month, followed by administration of a fourth dose about 10-13 months after administration of the first dose. In another embodiment, said schedule consists of a series of 3 doses, wherein each dose is separated by an interval of about 2 months, followed by administration of a fourth dose about 10-13 months after administration of the first dose.

In one embodiment, the multiple dose schedule includes administering at least one dose (e.g., 1, 2, or 3 doses) in the first year of life, followed by at least one dose when the child reaches toddlerhood.

In one embodiment, the multiple dose schedule consists of a series of 2 or 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 12-18 months of age. In one embodiment, the schedule consists of a series of 3 doses, wherein each dose is separated by an interval of from about 1 month to about 2 months (e.g., an interval of 28-56 days between doses), beginning at 2 months of age, followed by a dose at 12-15 months of age. In another embodiment, the schedule consists of a series of 2 doses, separated by an interval of about 2 months, beginning at 2 months of age, followed by a dose at 12-18 months of age.

In one embodiment, the multiple-dose schedule consists of a 4-dose vaccine series given at 2, 4, 6, and 12-15 months of age.

In one embodiment, the primary dose is administered on day 0 and one or more booster doses are administered at intervals of about 2 to about 24 weeks, preferably at a dosing interval of 4-8 weeks.

In one embodiment, a primary dose is administered on day 0 and a booster dose is administered approximately 3 months later.

In another embodiment, the present invention relates to a set of immunogenic compositions of the invention for use in parallel administration of a first and second immunogenic composition.

In one embodiment, the vaccination schedule for said concurrent administration is a single dose administration (the administration of the first and second immunogenic compositions, although in separate unit dosage forms, is considered a single dose for the purposes of determining the immunization schedule). In a particular embodiment, said single dose administration schedule is for healthy individuals at least 2 years of age.

In one embodiment, the vaccination schedule of said parallel administration is a multiple dose schedule, in particular any of the multiple dose schedules for parallel administration described above.

In one embodiment, the present invention relates to a set of immunogenic compositions as disclosed herein for use in the first and second immunogenic compositions.

In one embodiment, the first immunogenic composition of the invention is administered first, and the second immunogenic composition is administered second. In another embodiment, the second immunogenic composition is administered first, and the first immunogenic composition of the invention is administered second.

In one embodiment, the vaccination schedule of said sequential administration consists of a series of 2, 3, 4, 5, 6, 7 or 8 doses.

In one embodiment, the vaccination schedule for said sequential administration consists of a series of 2 doses. In one embodiment, the vaccination schedule for said sequential administration consists of a series of 4 doses. In one embodiment, the vaccination schedule for said sequential administration consists of a series of 6 doses. In one embodiment, the vaccination schedule for said sequential administration consists of a series of 8 doses.

In one embodiment, the vaccination schedule with said sequential administration consists of a series of 2 doses. In one embodiment, the vaccination schedule of said sequential administration consists of a series of 2 doses separated by an interval of from about 1 month to about 12 months. In a particular embodiment, said multiple dose administration schedule consists of a series of 2 doses separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said multiple dose administration schedule consists of a series of 2 doses separated by an interval of about 1 month. In a particular embodiment, said multiple dose administration schedule consists of a series of 2 doses separated by an interval of about 2 months.

In one embodiment of said two-dose schedule, the first immunogenic composition of the invention is administered first and the second immunogenic composition is administered second. In another embodiment, the second immunogenic composition is administered first and the first immunogenic composition of the invention is administered second.

In one embodiment, the vaccination schedule of said sequential administration consists of a series of 3 doses. In a specific embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of from about 1 to about 12 months. In a specific embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a specific embodiment, said schedule consists of a series of 3 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 3-dose schedule, the first immunogenic composition of the invention is administered in the first two doses and the second immunogenic composition is administered in the third dose.

In another embodiment of the three dose schedule, the second immunogenic composition is administered in the first two doses and the first immunogenic composition of the invention is administered in the third dose.

In another embodiment of said 3 dose administration schedule, the first immunogenic composition of the invention is administered as a first dose, the second immunogenic composition is administered as a second dose, and the first immunogenic composition according to the invention is administered as a third dose.

In another embodiment of the three-dose schedule, the second immunogenic composition is administered as a first dose, the first immunogenic composition of the invention is administered as a second dose, and the second immunogenic composition is administered as a third dose.

In another embodiment of the 3 dose schedule, the first immunogenic composition of the invention is administered as a first dose and the second immunogenic composition is administered as a second and third dose.

In another embodiment of the three dose schedule, the second immunogenic composition is administered as the first dose, and the first immunogenic composition of the invention is administered as the second and third doses.

In one embodiment, the vaccination schedule of said sequential administration consists of a 4-dose series.

In a particular embodiment, said schedule consists of a series of 4 doses, where each dose is separated by an interval of from about 1 to about 12 months. In a particular embodiment, said schedule consists of a series of 4 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said schedule consists of a series of 4 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 4 dose schedule, the first immunogenic composition of the invention is administered in the first three doses and the second immunogenic composition is administered in the fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first three doses, and the first immunogenic composition of the invention is administered as the fourth dose.

In another embodiment of the said 4 dose administration schedule, the first immunogenic composition of the invention is administered as the first and second doses, and the second immunogenic composition is administered as the third and fourth doses.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first and second doses, and the first immunogenic composition of the invention is administered as the third and fourth doses.

In another embodiment of said 4 dose administration schedule, the first immunogenic composition of the invention is administered as a first and second dose, the second immunogenic composition is administered as a third dose, and the first immunogenic composition is administered as a fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first and second doses, the first immunogenic composition of the invention is administered as the third dose, and the second immunogenic composition is administered as the fourth dose.

In another embodiment of the said 4 dose administration schedule, the first immunogenic composition of the invention is administered as a first dose, and the second immunogenic composition is administered as a second, third and fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first dose, and the first immunogenic composition of the invention is administered as the second, third and fourth doses.

In another embodiment of said 4 dose administration schedule, the first immunogenic composition of the invention is administered as a first dose, the second immunogenic composition is administered as a second dose, the first immunogenic composition of the invention is administered as a third dose, and the second immunogenic composition is administered as a fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as a first dose, the first immunogenic composition of the invention is administered as a second dose, the second immunogenic composition is administered as a third dose, and the first immunogenic composition is administered as a fourth dose.

In another embodiment of the said 4 dose administration schedule, the first immunogenic composition of the invention is administered as a first dose, the second immunogenic composition is administered as a second dose, and the first immunogenic composition of the invention is administered as a third and fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first dose, the first immunogenic composition of the invention is administered as the second dose, and the second immunogenic composition is administered as the third and fourth doses.

In another embodiment of the said 4 dose administration schedule, the first immunogenic composition of the invention is administered as a first dose, the second immunogenic composition is administered as a second and third dose, and the first immunogenic composition of the invention is administered as a fourth dose.

In another embodiment of the said 4 dose administration schedule, the second immunogenic composition is administered as the first dose, the first immunogenic composition of the invention is administered as the second and third doses, and the second immunogenic composition is administered as the fourth dose.

In one embodiment, the vaccination schedule of said sequential administration consists of a series of 5 doses.

In a particular embodiment, said schedule consists of a series of 5 doses, where each dose is separated by an interval of from about 1 to about 12 months. In a particular embodiment, said schedule consists of a series of 5 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said schedule consists of a series of 5 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 5-dose schedule, the first immunogenic composition (designated ^1st 1C in the table below) and the second immunogenic composition (designated ^2nd 1C in the table below) are administered in the following order:

The above table shows the order of administration of the first and second immunogenic compositions (designated ^1st 1C and ^2nd 1C, respectively) for different doses, for example, schedule No. 1 should be read as follows: in an embodiment of the said 5-dose administration schedule, the second immunogenic composition is administered as the first, second, third and fourth doses, and the first immunogenic composition of the invention is administered as the fifth dose.

In a preferred embodiment, the order of administration of the first and second immunogenic compositions corresponds to schedule 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, or 21.

In one embodiment, the vaccination schedule with said sequential dose consists of a series of 6 doses.

In a particular embodiment, said schedule consists of a series of 6 doses, where each dose administration is separated by an interval of from about 1 to about 12 months. In a particular embodiment, said schedule consists of a series of 6 doses, where each dose administration is separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said schedule consists of a series of 6 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 6 dose schedule, the first immunogenic composition and the second immunogenic composition are administered in the order of any of the 30 schedules provided for the 5 dose schedule (see Table 1 above, Schedules 1-30), followed by the sixth dose. In one embodiment, the first immunogenic composition of the invention is administered as the sixth dose. In another embodiment, the second immunogenic composition is administered as the sixth dose.

In an embodiment, the vaccination schedule with said sequential dose consists of a series of 7 doses.

In a specific embodiment, said regimen consists of a series of 7 doses, where each dose is separated by an interval of from about 1 to about 12 months. In a specific embodiment, said schedule consists of a series of 7 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a specific embodiment, said schedule consists of a series of 7 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 7 dose schedule, the first immunogenic composition and the second immunogenic composition are administered in the order of any of the schedules provided for the 6 dose schedule (see above), followed by the seventh dose. In one embodiment, the first immunogenic composition of the invention is administered as the seventh dose. In another embodiment, the second immunogenic composition is administered as the seventh dose.

In one embodiment, the vaccination schedule with said sequential dose consists of a series of 8 doses.

In a particular embodiment, said schedule consists of a series of 8 doses, where each dose is separated by an interval of from about 1 to about 12 months. In a particular embodiment, said schedule consists of a series of 8 doses, where each dose is separated by an interval of from about 1 month to about 6 months. In a particular embodiment, said schedule consists of a series of 8 doses, where each dose is separated by an interval of from about 1 to about 2 months.

In one embodiment of the 8-dose schedule, the first immunogenic composition of the invention and the second immunogenic composition are administered in the order of any of the schedules provided for the 7-dose schedule (see above), followed by the eighth dose. In one embodiment, the first immunogenic composition of the invention is administered as the eighth dose. In another embodiment, the second immunogenic composition is administered as the eighth dose.

In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular injection. In one embodiment, the immunogenic compositions disclosed herein are administered by subcutaneous injection.

In one embodiment, the immunogenic compositions are administered by intramuscular injection into the thigh or arm. In one embodiment, the injection site is the anterolateral thigh muscle or the deltoid muscle.

In one embodiment, the immunogenic compositions are administered by subcutaneous injection into the thigh or arm. In one embodiment, the injection site is the adipose tissue over the anterolateral thigh muscle or the adipose tissue over the triceps.

When administered concomitantly, the first injection may be given in one thigh and the second in the other thigh (preferably in the anterolateral thigh muscles). Alternatively, the first injection may be given in one arm and the second in the other arm (preferably in the deltoid muscles). The first injection may also be given in the thigh and the second in the arm, or the first injection may be given in the arm and the second in the thigh.

In one aspect, the invention relates to a set of immunogenic compositions of the present invention for use in any of the immunization schedules described above.

11. Kit and method

In one embodiment, the invention relates to a kit comprising an immunogenic composition or a set of immunogenic compositions disclosed herein and a package insert.

In one embodiment, the package insert discloses the ability of the composition or set of immunogenic compositions to induce the production of functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, said package insert indicates the ability of the composition or set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C at a concentration of ≥0.35 μg/ml in the human population.

In one embodiment, said package insert indicates the ability of the composition or set of immunogenic compositions to induce OPA titers in a human population against S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention and

- combining in one kit the said immunogenic composition and an information leaflet, which indicates the ability of the said composition to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the specified set of immunogenic compositions to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention and

- combining in one kit the said immunogenic composition and an information leaflet, which indicates the ability of the composition to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention and

- combining in one set the specified immunogenic composition and an information leaflet, which indicates the ability of the composition to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the set of immunogenic compositions to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention;

- printing of an information leaflet, which indicates the ability of the said composition to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified immunogenic composition and the specified information leaflet.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention;

- printing of an information leaflet, which indicates the ability of the specified set of immunogenic compositions to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified set of immunogenic compositions and the specified information leaflet.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention;

- printing of an information leaflet, which indicates the ability of the composition to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml;

- combining in one set the specified immunogenic composition and the specified information leaflet.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention;

- printing of an information leaflet, which indicates the ability of the set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml;

- combining in one set the specified set of immunogenic compositions and the specified information leaflet.

In one embodiment, the invention relates to a method for preparing a kit containing an immunogenic composition and an information leaflet, wherein said method comprises the steps of:

- obtaining an immunogenic composition according to the present invention;

- printing of an information leaflet, which indicates the ability of the composition to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C;

combining in one set the specified immunogenic composition and the specified information leaflet.

In one embodiment, the invention relates to a method for preparing a kit containing a set of immunogenic compositions and an information leaflet, wherein said method comprises the steps of:

- obtaining a set of immunogenic compositions according to the present invention;

- printing of an information leaflet, which indicates the ability of the set of immunogenic compositions to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified set of immunogenic compositions and the specified information leaflet.

12. The invention also provides the following embodiments, defined in the following numbered claims 1 to 361.

1. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15B, a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A.

2. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15B or 15C and a glycoconjugate of S. pneumoniae serotype 15A.

3. The immunogenic composition according to claim 2, wherein said composition does not contain the capsular saccharide of S. pneumoniae serotype 15C when the glycoconjugate of S. pneumoniae serotype 15B is present in the composition, and does not contain the capsular saccharide of S. pneumoniae serotype 15B when the glycoconjugate of S. pneumoniae serotype 15C is present.

4. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15A.

5. An immunogenic composition according to claim 4, wherein said composition does not contain the capsular saccharide of S. pneumoniae serotype 15C.

6. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15C and a glycoconjugate of S. pneumoniae serotype 15A.

7. An immunogenic composition according to claim 6, wherein said composition does not contain the capsular saccharide of S. pneumoniae serotype 15B.

8. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition additionally comprises at least one glycoconjugate of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and/or 23F.

9. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

10. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of each of the following ten S. pneumoniae serotypes: 1, 5, 4, 6B, 7F, 9V, 14, 18C, 19F and 23F.

11. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of each of the following twelve S. pneumoniae serotypes: 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

12. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of each of the following thirteen S. pneumoniae serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

13. An immunogenic composition according to any one of claims 1-12, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 22F.

14. An immunogenic composition according to any one of claims 1-13, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 33F.

15. An immunogenic composition according to any one of claims 1-14, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 8.

16. An immunogenic composition according to any one of claims 1-15, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 10A.

17. An immunogenic composition according to any one of claims 1-16, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 11A.

18. An immunogenic composition according to any one of claims 1-17, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 12F.

19. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of S. pneumoniae serotype 22F and at least one glycoconjugate of S. pneumoniae serotype 33F.

20. An immunogenic composition according to any one of claims 1-7, wherein said immunogenic composition further comprises at least one glycoconjugate of each of the following six S. pneumoniae serotypes: 22F, 33F, 12F, 10A, 11A and 8.

21. An immunogenic composition according to any one of claims 1-20, wherein all glycoconjugates are individually conjugated to a carrier protein.

22. An immunogenic composition according to any one of claims 1-21, wherein the S. pneumoniae serotype 15B glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

23. The immunogenic composition of any one of claims 1-22, wherein the S. pneumoniae serotype 15C glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

24. The immunogenic composition of any one of claims 1-23, wherein the S. pneumoniae serotype 15A glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

25. The immunogenic composition of any one of claims 1-24, wherein the S. pneumoniae serotype 22F glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

26. The immunogenic composition of any one of claims 1-25, wherein the S. pneumoniae serotype 33F glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

27. The immunogenic composition of any one of claims 1-26, wherein the S. pneumoniae serotype 12F glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

28. The immunogenic composition of any one of claims 1-27, wherein the S. pneumoniae serotype 10A glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

29. The immunogenic composition of any one of claims 1-28, wherein the S. pneumoniae serotype 11A glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

30. The immunogenic composition of any one of claims 1-29, wherein the S. pneumoniae serotype 8 glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

31. An immunogenic composition according to any one of claims 1-30, wherein the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, if present, are conjugated to CRM ₁₉₇ .

32. The immunogenic composition of any one of claims 1-31, wherein the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F, if present, are conjugated with CRM ₁₉₇ .

33. An immunogenic composition according to any one of claims 1-32, wherein the glycoconjugates of S. pneumoniae serotypes 6A and 19A, if present, are conjugated to CRM ₁₉₇ .

34. The immunogenic composition of any one of claims 1-33, wherein the glycoconjugate from S. pneumoniae serotype 3, if present, is conjugated to CRM ₁₉₇ .

35. An immunogenic composition according to any one of claims 1-34, wherein all glycoconjugates are individually conjugated to CRM ₁₉₇ .

36. The immunogenic composition of any one of claims 1-24, wherein the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F, if present, are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F is conjugated to DT.

37. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 8 different pneumococcal serotypes.

38. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 9 different pneumococcal serotypes.

39. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 10 different pneumococcal serotypes.

40. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 11 different pneumococcal serotypes.

41. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 12 different pneumococcal serotypes.

42. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 13 different pneumococcal serotypes.

43. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 14 different pneumococcal serotypes.

44. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 15 different pneumococcal serotypes.

45. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 16 different pneumococcal serotypes.

46. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 17 different pneumococcal serotypes.

47. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 18 different pneumococcal serotypes.

48. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 19 different pneumococcal serotypes.

49. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 20 different pneumococcal serotypes.

50. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 21 different pneumococcal serotypes.

51. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 22 different pneumococcal serotypes.

52. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates of 23 different pneumococcal serotypes.

53. An immunogenic composition according to any one of claims 1-36, containing glycoconjugates from 24 different pneumococcal serotypes.

54. An immunogenic composition according to any one of paragraphs 1-36, which is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugate composition.

55. An immunogenic composition according to any one of claims 1-36, which is a 16-valent pneumococcal conjugate composition.

56. An immunogenic composition according to any one of claims 1-36, which is a 19-valent pneumococcal conjugate composition.

57. An immunogenic composition according to any one of claims 1-36, which is a 20-valent pneumococcal conjugate composition.

58. An immunogenic composition according to any one of claims 1-36, which is a 21-valent pneumococcal conjugate composition.

59. An immunogenic composition according to any one of claims 1-36, which is a 21-valent pneumococcal conjugate composition.

60. An immunogenic composition according to any one of claims 1-36, which is a 22-valent pneumococcal conjugate composition.

61. An immunogenic composition according to any one of claims 1-36, which is a 23-valent pneumococcal conjugate composition.

62. An immunogenic composition according to any one of claims 1-36, which is a 24-valent pneumococcal conjugate composition.

63. An immunogenic composition according to any one of claims 1-63, wherein all glycoconjugates of the immunogenic composition are individually conjugated to a carrier protein.

64. An immunogenic composition according to any one of claims 1-64, wherein the glycoconjugates are individually conjugated to CRM ₁₉₇ .

65. An immunogenic composition according to any one of claims 1-64, wherein all glycoconjugates are individually conjugated to CRM ₁₉₇ .

66. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not contain a capsular saccharide of S. pneumoniae serotype 15B, and wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration.

67. A set of immunogenic compositions according to item 66 for parallel, concomitant or sequential administration.

68. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not contain a capsular saccharide of S. pneumoniae serotype 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

69. A set of immunogenic compositions according to item 68 for parallel, concomitant or sequential administration.

70. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

71. A set of immunogenic compositions according to item 70 for parallel, concomitant or sequential administration.

72. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not contain a capsular saccharide of S. pneumoniae serotype 15B, and wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration.

73. A set of immunogenic compositions according to item 72 for parallel, concomitant or sequential administration.

74. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said compositions do not contain a capsular saccharide of S. pneumoniae serotype 15C, and wherein said compositions are intended for simultaneous, parallel, concomitant or sequential administration.

75. A set of immunogenic compositions according to item 74 for parallel, concomitant or sequential administration.

76. A set of immunogenic compositions comprising: (a) a first immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B; and (b) a second immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic compositions are intended for simultaneous, parallel, concomitant or sequential administration.

77. A set of immunogenic compositions according to item 76 for parallel, concomitant or sequential administration.

78. A set of immunogenic compositions according to any one of claims 66-77, wherein said first immunogenic composition additionally contains, in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

79. A set of immunogenic compositions according to claim 78, wherein said first immunogenic composition additionally contains glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F.

80. A set of immunogenic compositions according to any one of claims 78-79, wherein said first immunogenic composition additionally contains glycoconjugates of S. pneumoniae serotypes 6A and 19A.

81. A set of immunogenic compositions according to any one of claims 78-80, wherein said first immunogenic composition additionally comprises a glycoconjugate of S. pneumoniae serotype 3.

82. A set of immunogenic compositions according to any one of claims 78-81, wherein said first immunogenic composition additionally comprises a glycoconjugate of S. pneumoniae serotype 22F.

83. A set of immunogenic compositions according to any one of claims 78-82, wherein said first immunogenic composition additionally comprises a glycoconjugate of S. pneumoniae serotype 33F.

84. A set of immunogenic compositions according to any one of claims 78-81, wherein said first immunogenic composition additionally contains, in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, glycoconjugates of S. pneumoniae serotypes 8, 10A, 11A, 12F, 22F and 33F.

85. A set of immunogenic compositions according to any one of claims 78-84, wherein all glycoconjugates of the first immunogenic composition are individually conjugated to a carrier protein.

86. A set of immunogenic compositions according to any one of claims 78-85, wherein the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F of the first immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

87. A set of immunogenic compositions according to any one of claims 78-86, wherein the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F of the first immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

88. A set of immunogenic compositions according to any one of claims 78-87, wherein the glycoconjugates of S. pneumoniae serotypes 6A and 19A of the first immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

89. A set of immunogenic compositions according to any one of claims 78-88, wherein the S. pneumoniae serotype 3 glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

90. A set of immunogenic compositions according to any one of claims 78-89, wherein the S. pneumoniae serotype 22F glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

91. A set of immunogenic compositions according to any one of claims 78-90, wherein the S. pneumoniae serotype 33F glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

92. A set of immunogenic compositions according to any one of claims 78-91, wherein the S. pneumoniae serotype 10A glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

93. A set of immunogenic compositions according to any one of claims 78-92, wherein the S. pneumoniae serotype HA glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

94. A set of immunogenic compositions according to any one of claims 78-93, wherein the S. pneumoniae serotype 12F glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

95. A set of immunogenic compositions according to any one of claims 78-94, wherein the S. pneumoniae serotype 8 glycoconjugate of the first immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

96. A set of immunogenic compositions according to any one of claims 78-95, wherein all glycoconjugates of the first immunogenic compositions are individually conjugated with CRM ₁₉₇ .

97. A set of immunogenic compositions according to any one of claims 78-85, wherein the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F of the first immunogenic composition, if present, are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C of the first immunogenic composition, if present, is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F of the first immunogenic composition, if present, is conjugated to DT.

98. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises from 7 to 25 different serotypes of S. pneumoniae.

99. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition contains glycoconjugates of 7 different serotypes.

100. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition contains glycoconjugates of 7 different serotypes.

101. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition contains glycoconjugates of 8 different serotypes.

102. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 10 different serotypes.

103. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 11 different serotypes.

104. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 12 different serotypes.

105. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 13 different serotypes.

106. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 14 different serotypes.

107. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 15 different serotypes.

108. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 16 different serotypes.

109. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 17 different serotypes.

110. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 18 different serotypes.

111. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 19 different serotypes.

112. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 20 different serotypes.

113. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition contains glycoconjugates of 21 different serotypes.

114. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 22 different serotypes.

115. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 23 different serotypes.

116. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition comprises glycoconjugates of 24 different serotypes.

117. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 7-valent pneumococcal conjugate composition.

118. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is an 8-valent pneumococcal conjugate composition.

119. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 9-valent pneumococcal conjugate composition.

120. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 10-valent pneumococcal conjugate composition.

121. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is an 11-valent pneumococcal conjugate composition.

122. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 12-valent pneumococcal conjugate composition.

123. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 13-valent pneumococcal conjugate composition.

124. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 14-valent pneumococcal conjugate composition.

125. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 15-valent pneumococcal conjugate composition.

126. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 16-valent pneumococcal conjugate composition.

127. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 17-valent pneumococcal conjugate composition.

128. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is an 18-valent pneumococcal conjugate composition.

129. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 19-valent pneumococcal conjugate composition.

130. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 20-valent pneumococcal conjugate composition.

131. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 21-valent pneumococcal conjugate composition.

132. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 22-valent pneumococcal conjugate composition.

133. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 23-valent pneumococcal conjugate composition.

134. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 24-valent pneumococcal conjugate composition.

135. A set of immunogenic compositions according to any one of claims 66-97, wherein the first immunogenic composition is a 25-valent pneumococcal conjugate composition.

136. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is an 8-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, individually conjugated with CRM ₁₉₇ .

137. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is a 14-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated with CRM ₁₉₇ .

138. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated with CRM ₁₉₇ .

139. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F, individually conjugated with CRM ₁₉₇ .

140. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated with CRM ₁₉₇ .

141. A set of immunogenic compositions according to any one of claims 66-85, wherein the first immunogenic composition is a 20-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15B or 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated with CRM ₁₉₇ .

142. A set of immunogenic compositions according to any one of claims 66-141, wherein the first immunogenic compositions further comprise one or more adjuvants as described in section 4.

143. A set of immunogenic compositions according to any one of claims 66-142, wherein the first immunogenic composition is formulated as described in section 5.

144. A set of immunogenic compositions according to any one of claims 66-143, wherein said second immunogenic composition, in addition to the glycoconjugate of S. pneumoniae serotype 15A, further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

145. A set of immunogenic compositions according to claim 144, wherein said second immunogenic composition additionally contains glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F.

14 6. A set of immunogenic compositions according to any one of paragraphs 14 4-145, wherein said second immunogenic composition additionally contains glycoconjugates of S. pneumoniae serotypes 6A and 19A.

147. A set of immunogenic compositions according to any one of claims 144-146, wherein said second immunogenic composition additionally comprises a glycoconjugate of S. pneumoniae serotype 3.

148. A set of immunogenic compositions according to any one of claims 144-147, wherein said second immunogenic composition further comprises a glycoconjugate of S. pneumoniae serotype 22F.

149. A set of immunogenic compositions according to any one of claims 144-148, wherein said second immunogenic composition further comprises a glycoconjugate of S. pneumoniae serotype 33F.

150. A set of immunogenic compositions according to any one of claims 66-149, wherein all glycoconjugates of the second immunogenic composition are individually conjugated to a carrier protein.

151. A set of immunogenic compositions according to any one of claims 66-150, wherein the glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F of the second immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

152. A set of immunogenic compositions according to any one of claims 66-151, wherein the glycoconjugates of S. pneumoniae serotypes 1, 5 and 7F of the second immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

153. A set of immunogenic compositions according to any one of claims 66-152, wherein the glycoconjugates of S. pneumoniae serotypes 6A and 19A of the second immunogenic composition, if present, are conjugated to CRM ₁₉₇ .

154. A set of immunogenic compositions according to any one of claims 66-153, wherein the S. pneumoniae serotype 3 glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

155. A set of immunogenic compositions according to any one of claims 66-154, wherein the S. pneumoniae serotype 22F glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

156. A set of immunogenic compositions according to any one of claims 66-155, wherein the S. pneumoniae serotype 33F glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

157. A set of immunogenic compositions according to any one of claims 66-156, wherein the S. pneumoniae serotype 10A glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

158. A set of immunogenic compositions according to any one of claims 66-157, wherein the S. pneumoniae serotype HA glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

159. A set of immunogenic compositions according to any one of claims 66-158, wherein the S. pneumoniae serotype 12F glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

160. A set of immunogenic compositions according to any one of claims 66-153, wherein the S. pneumoniae serotype 8 glycoconjugate of the second immunogenic composition, if present, is conjugated to CRM ₁₉₇ .

161. A set of immunogenic compositions according to any one of claims 66-160, wherein all glycoconjugates of the second immunogenic compositions are individually conjugated with CRM ₁₉₇ .

162. A set of immunogenic compositions according to any one of claims 66-150, wherein the glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F of the second immunogenic composition, if present, are individually conjugated to PD, the glycoconjugate of S. pneumoniae serotype 18C of the second immunogenic composition, if present, is conjugated to TT, and the glycoconjugate of S. pneumoniae serotype 19F of the second immunogenic composition, if present, is conjugated to DT.

163. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition comprises from 7 to 25 different serotypes of S. pneumoniae.

164. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition comprises from 7 to 20 different serotypes of S. pneumoniae.

165. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions contain glycoconjugates of 7 different serotypes.

166. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions contain glycoconjugates of 8 different serotypes.

167. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions comprise glycoconjugates of 10 different serotypes.

168. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions comprise glycoconjugates of 15 different serotypes.

169. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions comprise glycoconjugates of 16 different serotypes.

170. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic compositions comprise glycoconjugates of 20 different serotypes.

171. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-valent pneumococcal conjugate composition.

172. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 7-valent pneumococcal conjugate composition.

173. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is an 8-valent pneumococcal conjugate composition.

174. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 9-valent pneumococcal conjugate composition.

175. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 10-valent pneumococcal conjugate composition.

176. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is an 11-valent pneumococcal conjugate composition.

177. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 12-valent pneumococcal conjugate composition.

178. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 13-valent pneumococcal conjugate composition.

179. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 14-valent pneumococcal conjugate composition.

180. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 15-valent pneumococcal conjugate composition.

181. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 16-valent pneumococcal conjugate composition.

182. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 17-valent pneumococcal conjugate composition.

183. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is an 18-valent pneumococcal conjugate composition.

184. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 19-valent pneumococcal conjugate composition.

185. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 20-valent pneumococcal conjugate composition.

186. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 21-valent pneumococcal conjugate composition.

187. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 22-valent pneumococcal conjugate composition.

188. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 23-valent pneumococcal conjugate composition.

189. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 24-valent pneumococcal conjugate composition.

190. A set of immunogenic compositions according to any one of claims 66-162, wherein the second immunogenic composition is a 25-valent pneumococcal conjugate composition.

191. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is an 8-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, individually conjugated with CRM ₁₉₇ .

192. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 14-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated with CRM ₁₉₇ .

193. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated with CRM ₁₉₇ .

194. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F, individually conjugated with CRM ₁₉₇ .

195. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated with CRM ₁₉₇ .

196. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 20-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A, said composition further comprises glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated to CRM ₁₉₇ .

197. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 9-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, individually conjugated with CRM ₁₉₇ .

198. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 15-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually conjugated with CRM ₁₉₇ .

199. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 22F, individually conjugated with CRM ₁₉₇ .

200. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 16-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F and 33F, individually conjugated with CRM ₁₉₇ .

201. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 17-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated with CRM ₁₉₇ .

202. A set of immunogenic compositions according to any one of claims 66-143, wherein the second immunogenic composition is a 21-valent pneumococcal conjugate composition, wherein in addition to the glycoconjugate of S. pneumoniae serotype 15A and the glycoconjugate of S. pneumoniae serotype 15C, said composition further comprises glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, IF, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 23F, 22F and 33F, individually conjugated with CRM ₁₉₇ .

203. A set of immunogenic compositions according to any one of claims 66-202, wherein the second immunogenic compositions further comprise one or more adjuvants as described in section 4.

204. A set of immunogenic compositions according to any one of claims 66-203, wherein the second immunogenic composition is formulated as described in section 5.

205. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 15B and 15C.

206. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 15A and 15C.

207. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 15A and 15B.

208. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain a glycoconjugate of S. pneumoniae serotype 15B.

209. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain a glycoconjugate of S. pneumoniae serotype 15C.

210. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain a glycoconjugate of S. pneumoniae serotype 15A.

211. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15B, 15C, 18C, 19F and 23F.

212. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 15C, 18C, 19F and 23F.

213. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 15B, 18C, 19F and 23F.

214. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15B, 18C, 19F and 23F.

215. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15C, 18C, 19F and 23F.

216. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 4, 6B, 9V, 14, 15A, 18C, 19F and 23F.

217. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19F and 23F.

218. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19F and 23F.

219. An immunogenic composition containing a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19F and 23F.

220. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15B, 18C, 19F and 23F.

221. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15C, 18C, 19F and 23F.

222. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 15A, 18C, 19F and 23F.

223. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F and 23F.

224. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F and 23F.

225. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F and 23F.

226. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F.

227. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F and 23F.

228. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F and 23F.

229. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F and 23F.

230. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F and 23F.

231. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F and 23F.

232. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F.

233. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F and 23F.

234. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F and 23F.

235. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

236. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

237. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

238. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

23 9. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

240. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

241. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

242. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

243. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

244. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

245. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

24 6. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

247. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

248. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

249. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

250. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

251. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

252. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

253. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

254. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

255. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

256. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

257. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

258. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

259. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F.

260. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not contain glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23F and 33F.

261. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15A, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

262. An immunogenic composition comprising a glycoconjugate of S. pneumoniae serotype 15C, wherein said immunogenic composition does not comprise glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

263. An immunogenic composition according to any one of claims 205-262, wherein the glycoconjugates are individually conjugated to a carrier protein.

264. The immunogenic composition of any one of claims 205-263, wherein the S. pneumoniae serotype 15C glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

265. The immunogenic composition of any one of claims 205-264, wherein the S. pneumoniae serotype 15A glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

266. The immunogenic composition of any one of claims 205-265, wherein the S. pneumoniae serotype 15B glycoconjugate, if present, is conjugated to CRM ₁₉₇ .

267. An immunogenic composition according to any one of claims 205-266, containing glycoconjugates of 2 to 25 different serotypes of S. pneumoniae.

268. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 2 different pneumococcal serotypes.

269. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 3 different pneumococcal serotypes.

270. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 7 different pneumococcal serotypes.

271. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 8 different pneumococcal serotypes.

272. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 9 different pneumococcal serotypes.

273. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 10 different pneumococcal serotypes.

274. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 11 different pneumococcal serotypes.

275. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 12 different pneumococcal serotypes.

27 6. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 13 different pneumococcal serotypes.

277. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 14 different pneumococcal serotypes.

278. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 15 different pneumococcal serotypes.

279. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 16 different pneumococcal serotypes.

280. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 17 different pneumococcal serotypes.

281. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 18 different pneumococcal serotypes.

282. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 19 different pneumococcal serotypes.

283. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 19 different pneumococcal serotypes.

284. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 19 different pneumococcal serotypes.

285. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 20 different pneumococcal serotypes.

286. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 21 different pneumococcal serotypes.

287. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 22 different pneumococcal serotypes.

288. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 23 different pneumococcal serotypes.

289. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 24 different pneumococcal serotypes.

290. An immunogenic composition according to any one of claims 205-267, containing glycoconjugates of 25 different pneumococcal serotypes.

291. An immunogenic composition according to any one of paragraphs 205-267, which is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugate composition.

292. An immunogenic composition according to any one of claims 205-267, which is a bivalent pneumococcal conjugate composition.

293. An immunogenic composition according to any one of claims 205-267, which is a trivalent pneumococcal conjugate composition.

294. An immunogenic composition according to any one of claims 205-267, which is a 4-valent pneumococcal conjugate composition.

295. An immunogenic composition according to any one of claims 205-267, which is a 5-valent pneumococcal conjugate composition.

296. An immunogenic composition according to any one of claims 205-267, which is a 6-valent pneumococcal conjugate composition.

297. An immunogenic composition according to any one of claims 205-267, which is a 7-valent pneumococcal conjugate composition.

298. An immunogenic composition according to any one of claims 205-267, which is an 8-valent pneumococcal conjugate composition.

299. An immunogenic composition according to any one of claims 205-267, which is a 9-valent pneumococcal conjugate composition.

300. An immunogenic composition according to any one of claims 205-267, which is a 10-valent pneumococcal conjugate composition.

301. An immunogenic composition according to any one of claims 205-267, which is an 11-valent pneumococcal conjugate composition.

302. An immunogenic composition according to any one of paragraphs 205-267, which is a 12-valent pneumococcal conjugate composition.

303. An immunogenic composition according to any one of paragraphs 205-267, which is a 13-valent pneumococcal conjugate composition.

304. An immunogenic composition according to any one of paragraphs 205-267, which is a 14-valent pneumococcal conjugate composition.

305. An immunogenic composition according to any one of claims 205-267, which is a 15-valent pneumococcal conjugate composition.

306. An immunogenic composition according to any one of paragraphs 205-267, which is a 16-valent pneumococcal conjugate composition.

307. An immunogenic composition according to any one of claims 205-267, which is a 17-valent pneumococcal conjugate composition.

308. An immunogenic composition according to any one of claims 205-267, which is an 18-valent pneumococcal conjugate composition.

309. An immunogenic composition according to any one of claims 205-267, which is a 19-valent pneumococcal conjugate composition.

310. An immunogenic composition according to any one of paragraphs 205-267, which is a 20-valent pneumococcal conjugate composition.

311. An immunogenic composition according to any one of paragraphs 205-267, which is a 21-valent pneumococcal conjugate composition.

312. An immunogenic composition according to any one of paragraphs 205-267, which is a 22-valent pneumococcal conjugate composition.

313. An immunogenic composition according to any one of paragraphs 205-267, which is a 23-valent pneumococcal conjugate composition.

314. An immunogenic composition according to any one of paragraphs 205-267, which is a 24-valent pneumococcal conjugate composition.

315. An immunogenic composition according to any one of claims 205-314, wherein all glycoconjugates are individually conjugated to a carrier protein.

316. An immunogenic composition according to any one of claims 205-314, wherein the glycoconjugates are individually conjugated to CRM ₁₉₇ .

317. An immunogenic composition according to any one of claims 205-314, wherein all glycoconjugates are individually conjugated to CRM ₁₉₇ .

318. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

319. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 10-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

320. An immunogenic composition for use according to claim 305, wherein said glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F are conjugated with PD, said glycoconjugate of S. pneumoniae serotype 18C is conjugated with TT, and said glycoconjugate of S. pneumoniae serotype 19F is conjugated with DT.

321. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

322. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 12-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated with CRM ₁₉₇ .

323. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

324. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 13-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM ₁₉₇ .

325. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

326. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 14-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM ₁₉₇ .

327. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

328. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 15-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM ₁₉₇ .

329. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

330. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 19-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM ₁₉₇ .

331. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a vaccine that contains glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

332. An immunogenic composition according to any one of claims 205-317 for use in a method of supplementing a 20-valent pneumococcal conjugate vaccine, wherein said conjugates consist of glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

333. An immunogenic composition for use according to claim 318, wherein said glycoconjugates of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F conjugated with CRM ₁₉₇ .

334. An immunogenic composition according to any one of claims 210, 216, 222, 228, 234, 240, 246, 252 or 260 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15A but does not contain glycoconjugates of S. pneumoniae serotypes 15B and 15C.

335. An immunogenic composition according to any one of claims 208, 214, 220, 226, 232, 238, 244, 250 or 258 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15B but does not contain glycoconjugates of S. pneumoniae serotypes 15A and 15C.

336. An immunogenic composition according to any one of claims 209, 215, 221, 227, 233, 239, 245, 251 or 259 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15C but does not contain glycoconjugates of S. pneumoniae serotypes 15A and 15B.

337. An immunogenic composition according to any one of claims 207, 213, 219, 225, 231, 237, 243, 249, 254, 257 or 262 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15B, but does not contain a glycoconjugate of S. pneumoniae serotype 15C.

338. An immunogenic composition according to any one of claims 207, 213, 219, 225, 231, 237, 243, 249, 254, 257 or 262 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15A but does not contain a glycoconjugate of S. pneumoniae serotype 15C.

339. An immunogenic composition according to any one of claims 207, 213, 219, 225, 231, 237, 243, 249, 254, 257 or 262 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15B but does not contain a glycoconjugate of S. pneumoniae serotype 15C.

340. An immunogenic composition according to any one of claims 206, 212, 218, 224, 230, 236, 242, 248 or 256 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15A and a glycoconjugate of S. pneumoniae serotype 15C, but does not contain a glycoconjugate of S. pneumoniae serotype 15B.

341. An immunogenic composition according to any one of claims 206, 212, 218, 224, 230, 236, 242, 248 or 256 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15A but does not contain a glycoconjugate of S. pneumoniae serotype 15B.

342. An immunogenic composition according to any one of claims 206, 212, 218, 224, 230, 236, 242, 248 or 256 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15C but does not contain a glycoconjugate of S. pneumoniae serotype 15B.

343. An immunogenic composition according to any one of claims 205, 211, 217, 223, 229, 235, 241, 247, 253, 255 or 261 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15B and a glycoconjugate of S. pneumoniae serotype 15C, but does not contain a glycoconjugate of S. pneumoniae serotype 15A.

344. An immunogenic composition according to any one of claims 205, 211, 217, 223, 229, 235, 241, 247, 253, 255 or 261 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15B but does not contain a glycoconjugate of S. pneumoniae serotype 15A.

345. An immunogenic composition according to any one of claims 205, 211, 217, 223, 229, 235, 241, 247, 253, 255 or 261 for use in a method of supplementing a pneumococcal conjugate vaccine that contains a glycoconjugate of S. pneumoniae serotype 15C but does not contain a glycoconjugate of S. pneumoniae serotype 15A.

34 6. A kit containing an immunogenic composition according to any one of claims 1-65 or a set of immunogenic compositions according to any one of claims 66-204 and an information leaflet.

347. The kit according to item 346, wherein the said information leaflet indicates the ability of the composition or set of immunogenic compositions to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

348. The kit according to item 346 or item 347, wherein the said information leaflet indicates the ability of the composition or set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml.

349. A kit according to any one of paragraphs 346-348, wherein said package insert indicates the ability of the composition or set of immunogenic compositions to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C.

350. A method for preparing a kit containing an immunogenic composition and an information leaflet, comprising the steps of:

- obtaining an immunogenic composition according to any one of paragraphs 1-65 and

- combining in one kit the said immunogenic composition and an information leaflet, which indicates the ability of the said composition to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

351. A method for preparing a kit containing a set of immunogenic compositions according to any one of paragraphs 66-204 and an information leaflet, wherein said method includes the steps of:

- obtaining a set of immunogenic compositions and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the specified set of immunogenic compositions to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C.

352. A method for preparing a kit containing an immunogenic composition according to any one of paragraphs 1-65 and an information leaflet, wherein said method includes the steps of:

- obtaining an immunogenic composition according to any one of paragraphs 1-65 and

- combining in one kit the said immunogenic composition and an information leaflet, which indicates the ability of the composition to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml.

353. A method for preparing a kit containing a set of immunogenic compositions according to any one of paragraphs 66-204 and an information leaflet, wherein said method includes the steps of:

- obtaining a set of immunogenic compositions according to any of paragraphs 66-204 and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of ≥0.35 μg/ml.

354. A method for preparing a kit containing an immunogenic composition according to any one of paragraphs 1-65 and an information leaflet, wherein said method includes the steps of:

- obtaining an immunogenic composition according to any one of paragraphs 1-65 and

- combining in one set the specified immunogenic composition and an information leaflet, which indicates the ability of the composition to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C.

355. A method for preparing a kit containing a set of immunogenic compositions according to any one of paragraphs 66-204 and an information leaflet, wherein said method includes the steps of:

- obtaining a set of immunogenic compositions according to any of paragraphs 66-204 and

- combining in one kit the specified set of immunogenic compositions and an information leaflet, which indicates the ability of the set of immunogenic compositions to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C.

356. A method for preparing a kit containing an immunogenic composition and an information leaflet, comprising the following steps:

- obtaining an immunogenic composition according to any one of paragraphs 1-65;

- printing of an information leaflet, which indicates the ability of the said composition to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified immunogenic composition and the specified information leaflet.

357. A method for preparing a kit containing a set of immunogenic compositions and an information leaflet, comprising the steps of:

- obtaining a set of immunogenic compositions according to any of paragraphs 66-204;

- printing of an information leaflet, which indicates the ability of the specified set of immunogenic compositions to induce functional antibodies to S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified set of immunogenic compositions and the specified information leaflet.

358. A method for preparing a kit containing an immunogenic composition and an information leaflet, comprising the following steps:

- obtaining an immunogenic composition according to any one of paragraphs 1-65;

- printing of an information leaflet, which indicates the ability of the composition to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of> 0.35 μg/ml;

- combining in one set the specified immunogenic composition and the specified information leaflet.

359. A method for preparing a kit containing a set of immunogenic compositions and an information leaflet, comprising the following steps:

- obtaining a set of immunogenic compositions according to any of paragraphs 66-204;

- printing of an information leaflet, which indicates the ability of the set of immunogenic compositions to induce anticapsular antibodies to S. pneumoniae serotypes 15A, 15B and 15C in the human population at a concentration of> 0.35 μg/ml;

- combining in one set the specified set of immunogenic compositions and the specified information leaflet.

360. A method for preparing a kit containing an immunogenic composition and an information leaflet, comprising the steps of:

- obtaining an immunogenic composition according to any one of paragraphs 1-65;

- printing of an information leaflet, which indicates the ability of the composition to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C;

- combining in one set the specified immunogenic composition and the specified information leaflet.

361. A method for preparing a kit containing a set of immunogenic compositions and an information leaflet, comprising the following steps:

- obtaining a set of immunogenic compositions according to any of paragraphs 1-65;

- printing of an information leaflet, which indicates the ability of a set of immunogenic compositions to induce OPA titers in the human population against S. pneumoniae serotypes 15A, 15B and 15C;

combining in one set the specified set of immunogenic compositions and the specified information leaflet.

As used herein, the term "about" means within a statistically significant range of a value, 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 within 1% of the specified value or range. Sometimes, such a range may be within the experimental error typical of standard methods used to measure and/or determine the specified value or range. The tolerance encompassed by the term "about" will depend on the particular system being studied and can be readily appreciated by one of skill in the art. Whenever a range is specified in this application, each integer within that range is also considered an embodiment of the invention.

The terms "comprising", "comprise" and "comprises" in this document, as intended by the inventors, may in each case be optionally replaced by the terms "consisting of", "consist of" and "consists of", respectively.

All references or patent applications cited in this patent specification are incorporated herein by reference.

The invention is illustrated in the accompanying examples. The examples below are carried out using standard methods that are well known and routine to those skilled in the art, except where otherwise described in detail. The examples illustrate but do not limit the invention.

EXAMPLE

Example 1. Evaluation of cross-reactive opsonophagocytic immune responses within serogroup 15 Streptococcus pneumoniae

Materials and methods

Serum

Sera obtained from adult subjects,

vaccinated with the 20-valent pneumococcal conjugate vaccine (20vPnC) were tested by OPA assay for serotypes 15B, 15C and 15A.

Sera were collected during US clinical studies B7471001 (N=33, 1 month post-vaccination) and B7471002 (N=84, same pre- and post-vaccination), respectively.

Study B7471001 (ClinicalTrials.gov identifier: NCT02955160) was a randomized, controlled, single-blinded, 2-arm, parallel-group human study. Healthy adults aged 18–49 years received either a single intramuscular dose of 20-valent pneumococcal conjugate vaccine or a licensed tetanus, diphtheria, and pertussis (Tdap) combination vaccine (control group) (see https://clinicaltrials.gov/ct2/show/NCT02 955160, accessed September 22, 2020).

Study B7471002 (ClinicalTrials.gov identifier: NCT03313037) was a randomized, double-blind, parallel-group, phase 2 study. Healthy adults aged 60 to 64 years received either a single intramuscular dose of 20-valent pneumococcal conjugate vaccine followed by a saline dose 1 month later or Prevnar 13 (13vPnC) followed by a PPSV23 dose 1 month later (control group) (see https://clinicaltrials.gov/ct2/show/NCT03313037, accessed September 22, 2020).

The 20-valent pneumococcal conjugate vaccine (20vPnC) tested in these studies contained conjugates of pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F individually conjugated to diphtheria toxin-reactive carrier protein 197 (CRM ₁₉₇ ).

The 13-valent pneumococcal conjugate vaccine (13vPnC) tested contained conjugates of pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to diphtheria toxin-reactive carrier protein 197 (CRM ₁₉₇ ) (Prevenar 13®).

Microcolony Opsonophagocytic Activity (MCOPA) Assay Procedure

Opsonophagocytic assays (OPAs) are used to measure functional antibodies to S. pneumoniae serotypes 15A, 15B, and 15C in human serum. Test serum is added to assay reactions that measure the ability of capsular polysaccharide-specific immunoglobulin to opsonize bacteria, trigger complement deposition, and thereby promote phagocytosis and killing of bacteria by phagocytes. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial counts compared with control wells without test serum. The OPA titer is interpolated from two dilutions that include this 50% kill cutoff.

The OPA procedures are based on the methods described in Ni et al., (2005) Clin Diagn Lab Immunol 12:287–295.

The microcolony opsonophagocytic assay quantifies functional antibodies to S. pneumoniae by measuring bacterial survival in microcolony OPA reaction mixtures (mCOPA) containing the test serum. Serial dilutions (2.5-fold) of heat-inactivated serum were added to target bacteria in assay plates and incubated for 30 min with shaking. Rabbit complement (3-4 weeks old, final concentration 12.5%) and differentiated HL-60 cells were then added to each well at an approximate effector to target ratio of 200:1 and incubated at 37°C with shaking. To stop the reaction, 80 μl of 0.9% NaCl were added to all wells, the reaction solution was mixed, and a 10 μl aliquot was transferred to the wells of Millipore MultiScreenHTS HV filter plates containing 200 μl of water. The liquid from the plates was filtered under vacuum, 150 μl of HySoy medium was added to each well and filtered.

After filtration, the filter plates were incubated overnight in a 5% _CO2 incubator at 37°C and then fixed with Destain (Bio-Rad). The plates were then stained with Coomassie blue and destained once. Colonies were visualized and counted on a Cellular Technology Limited (CTL) IminunoSpot® analyzer. The OPA antibody titer was defined as the reciprocal of the lowest serum dilution that resulted in a 50% reduction in the number of bacterial colonies compared with control wells containing bacteria, effector cells, and complement but no serum.

Example 2. Cross-functional OPA against Streptococcus pneumoniae serotypes 15B, 15C, 15A

Cross-reactive antibodies that have functional, such as opsonophagocytic, activity are called cross-functional. Cross-functional reactions between Streptococcus pneumoniae serotypes 15A, 15B, and 15C were assessed. Opsonophagocytic assays were developed against serotype 15A, serotype 15B, and serotype 15C strains. Evaluation of sera from clinical trials B7471001 and B7471002 (see Example 1) showed that immunization with a vaccine containing 15B capsular polysaccharide conjugate induced strong cross-functional responses against serotype 15C isolates but only minor cross-functional responses against serotype 15A.

Results - OPA answers to 15B, 15C and 15A

The cross-functional response of immune sera obtained from adult subjects immunized with either 20vPnC or Tdap against serotypes 15B, 15C, and 15A was assessed in the respective microcolony opsonophagocytic activity (mcOPA) assays along with the homologous functional response to serotype 15B.

A panel of sera obtained from adult subjects immunized with 20vPnC or Tdap (study B7471001 (see above)) were assessed for OPA in relation to the homologous response to serotype 15B and for cross-reactivity of anti-15B antibodies to serotypes 15C and 15A.

As shown in Table 1, sera from subjects immunized with 20vPnC were able to kill both 15C strains. Sera from subjects immunized with 20vPnC were able to kill some of both 15A strains, but the titers were low.

Example 3. Cross-functional response to Streptococcus pneumoniae serotypes 15B and 15C

To confirm the results observed in Example 2 with a larger set of samples, cross-functional 15C OPAs were performed using sera obtained from subjects vaccinated with 20vPnC containing 15B capsular polysaccharide conjugate or 13vPnC without 15B capsular polysaccharide conjugate. GMT and 95% correlation coefficients were determined for the two study groups. GMR (geometric mean ratio) was also calculated to compare the two groups. The GMT for 15C OPA in the 20vPnC group was 164 and in the 13vPnC group was 14 (Table 2). The GMR between groups was 11.9 (Table 2), indicating that 15V-containing 20vPnC elicited cross-functional immune responses to 15C. The 15C OPA titer inverse cumulative distribution curves (RCDCs) also differed between the 20vPnC and 13vPnC groups (Fig. 1).

Abbreviations: GMR = geometric mean ratio; GMT = geometric mean titer; OPA = opsonophagocytic activity.

a. n = number of subjects with a given OPA titer for a given serotype.

b. GMT was calculated considering all subjects with available data for a given blood draw.

c. CIs are the inverse transformation of CIs based on the Student's t distribution for the mean log titers.

d. The GMT ratio (20vPnC/saline to 13vPnC/PPSV23) was calculated by back-transforming the mean difference in GMT values between vaccine sequences to a logarithmic scale.

e. The CI for this ratio is the inverse transformation of the CI based on the Student's t distribution for the mean difference in the logarithms of the measurements (2OvPnC/saline minus 13vPnC/PPSV23).

Conclusion

Well-characterized OPAs were developed for two Streptococcus pneumoniae serotype 15A strains and two S. pneumoniae serotype 15C strains. Sera from subjects immunized with the 20vPnC vaccine containing the 15B capsular polysaccharide showed cross-functional activity of the OPAs against both 15C strains. This cross-functional activity was confirmed in a larger cohort against a single 15C strain. Sera from subjects immunized with 20vPnC showed some cross-functional activity against the S. pneumoniae 15A strains tested, although the titers were low.

All publications and patent applications mentioned in the specification are representative of the level of skill in the art to which this invention pertains. All publications and patent applications are hereby 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.

Although the present invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, appropriate changes and modifications may be made in practice within the scope of the appended claims.

--->

SEQUENCE LISTS

<110> Pfizer Inc.

<120> IMMUNOGENIC COMPOSITIONS FOR USE IN PNEUMOCOCCAL VACCINES

<130> PC72510

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 1

tcgtcgtttt tcggtgcttt t 21

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 2

tcgtcgtttt tcggtcgttt t 21

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 3

tcgtcgtttt gtcgttttgt cgtt 24

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 4

tcgtcgtttc gtcgttttgt cgtt 24

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 5

tcgtcgtttt gtcgtttttt tcga 24

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 6

tcgtcgtttt tcggtgcttt t 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 7

tcgtcgtttt tcggtcgttt t 21

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 8

tcgtcgtttt gtcgttttgt cgtt 24

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 9

tcgtcgtttc gtcgttttgt cgtt 24

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> "Class B" CpG oligonucleotide

<400> 10

tcgtcgtttt gtcgtttttt tcga 24

<---

Claims (15)

1. A method for inducing an immune response in a human subject against S. pneumoniae serotypes 15B and 15C, comprising administering to said subject an immunogenic composition comprising an immunogenically effective amount of a glycoconjugate comprising a carrier protein and a capsular saccharide from S. pneumoniae serotype 15B, wherein said composition does not contain a capsular saccharide from S. pneumoniae serotype 15C. 2. The method according to claim 1, wherein said immunogenic composition additionally contains glycoconjugates containing a carrier protein and a capsular saccharide from a group of S. pneumoniae serotypes consisting of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 22F, 23F and 33F. 3. The method according to claim 2, wherein said immunogenic composition additionally contains glycoconjugates containing a carrier protein and a capsular saccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 22F, 23F and 33F. 4. The method according to any one of claims 1-3, wherein said immunogenic composition is at least a 20-valent pneumococcal conjugate composition. 5. The method according to any one of claims 1-4, wherein said carrier protein is selected from the group consisting of DT, TT, SCP, PD and CRM197. 6. The method of claim 5, wherein said carrier protein is CRM197. 7. The method according to any one of claims 1-6, wherein the carrier protein of all glycoconjugates is CRM197. 8. The method of any one of claims 1-7, wherein said method is effective for inducing in said subject the production of functional reactive antibodies against S. pneumoniae serotypes 15B and 15C. 9. The method according to any one of claims 1-8, wherein said method is effective for the prevention of a disease associated with serotypes 15B or 15C of S. pneumoniae. 10. The method according to any one of claims 1-9, wherein said method is effective for the prevention of a disease associated with serotypes 15B and 15C of S. pneumoniae. 11. The method of any one of claims 1-10, wherein the immunogenically effective amount of said glycoconjugate comprises approximately 2 μg of capsular saccharide from S. pneumoniae serotype 15B. 12. The method according to any one of claims 1-10, wherein the immunogenically effective amount of said glycoconjugate comprises from 2 μg to 5 μg of capsular saccharide from serotype 15B S. pneumoniae. 13. The method according to any one of claims 1-12, wherein said immunogenic composition does not contain a capsular saccharide from serotype 15A of S. pneumoniae. 14. The method of claim 13, wherein said method is effective for inducing in said subject the production of functional reactive antibodies against serotype 15A of S. pneumoniae. 15. The method of any one of claims 1-14, wherein said method is effective to increase the opsonophagocytic activity geometric mean titer (OPA GMT) against S. pneumoniae serotypes 15B and 15C by at least 100 times compared to the level before administration of said immunogenic composition.