Pneumococcal polysaccharide compositions and uses thereof

Abstract

The present disclosure provides compositions comprising purified capsular polysaccharides having one or more repeat units of Formula I: wherein each R is independently selected from H and -C(O)-CH3; and uses thereof.

Description

Background Technology

[0001] Streptococcus pneumoniae infection can cause invasive diseases in humans, such as meningitis, sepsis, and pneumonia. Redin et al., Microbiol. Spectr., Nov-December 2021; 9(3): e01150-21 (December 8, 2021). Pneumococcal disease is the cause of death in approximately 500,000 children under the age of 5 each year and poses a significant risk to the elderly and immunocompromised individuals. Takano et al., Sci. Rep., 9, 19823 (2019). Streptococcus pneumoniae produces structurally diverse capsular polysaccharides, a major virulence factor. Redin et al., Microbiol. Spectr., Nov-December 2021; 9(3): e01150-21 (December 8, 2021). To date, more than 100 Streptococcus pneumoniae serotypes have been described. Ganaie et al., mBio., May–June 2020; 11(3): e00937-20 (May 19, 2020). Summary of the Invention

[0002] In recent years, pneumococcal polysaccharide vaccines (PPV) and pneumococcal conjugate vaccines (PCV) targeting various pneumococcal serotypes have been developed and can be widely used for the prevention of pneumococcal disease. However, to date, vaccines targeting multiple serotypes, including serotype 38, have not been developed. Streptococcus pneumoniae The structure of the repeating unit of the serotype capsular polysaccharide is still unknown. Among other things, this disclosure provides a previously uncharacterized [structure / structure]. Streptococcus pneumoniae This document outlines research insights and technical solutions related to the discovery and characterization of serotype 38. In some embodiments, the research insights and technical solutions provided herein are used to provide immunogenic compositions (e.g., vaccines) with enhanced immunogenicity to offer protection against certain diseases, conditions, and illnesses, such as those targeting specific diseases. lung Streptococcus inflammatoryae Those related to serotype 38.

[0003] In some embodiments, this disclosure provides a composition comprising purified capsular polysaccharides having repeating units of Formula I: I

[0004] Each R is independently selected from H and –C(O)-CH3.

[0005] In some embodiments, about 60 to about 75% of the repeating units of the purified polysaccharide composition of Formula I are in α-Gal fThe residue contains an O-acetylated group at C2 (i.e., R is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains 0% to 100% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R is –C(O)-CH3 in Formula I). ​​In some embodiments, the purified capsular polysaccharide described herein contains greater than or equal to 60% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains less than 60% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains approximately 68% α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R is –C(O)-CH3 in Formula I).

[0006] In some embodiments, this disclosure provides a purified capsular polysaccharide, activated with a chemical reagent to generate reactive groups for conjugation with suitable conjugates (e.g., linkers or carrier proteins). In some embodiments, activation occurs in β-Gal f At residues C2, C3, C5, or C6 (i.e., at the position designated as R in Formula I above). In some embodiments, activation occurs in less than 100% of the repeating units in the purified capsular polysaccharide of Formula I. In some embodiments, activation occurs in about 1% to about 50% of the repeating units in the purified capsular polysaccharide of Formula I.

[0007] In some embodiments, this disclosure provides an immunogenic composition comprising (i) a purified capsular polysaccharide; and (ii) a polypeptide, wherein the purified capsular polysaccharide has repeating units of Formula I: I And each of the Rs is independently selected from H and –C(O)-CH3.

[0008] In some embodiments, this disclosure provides an induction method for treating Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae A method for an immune response to capsular polysaccharides, comprising administering to a subject an immunogenic composition comprising (i) a purified capsular polysaccharide; and (ii) a polypeptide, wherein the purified capsular polysaccharide has repeating units of Formula I: I And each of the Rs is independently selected from H and –C(O)-CH3.

[0009] In some embodiments, the immunogenic compositions described herein are based on the MAPS technology described in PCT patent applications PCT / US / 2012037412, PCT / US2019 / 050907, PCT / US2022 / 042964 and PCT / US2022 / 043156, the entire contents of each of which are incorporated herein by reference.

[0010] In some embodiments, the immunogenic compositions described herein comprise a purified polysaccharide composition of Formula I conjugated with a polypeptide. In some embodiments, the immunogenic compositions described herein comprise a purified polysaccharide composition of Formula I non-covalently complexed with a polypeptide.

[0011] In some embodiments, this disclosure provides a method for characterizing an immunogenic composition comprising purified capsular polysaccharide having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, and the method comprises the following steps: characterizing a plurality of units of formula I in the purified capsular polysaccharide, wherein R is –C(O)-CH3; and comparing the number of units of formula I (where R is –C(O)-CH3) with a reference standard.

[0012] In some embodiments, this disclosure provides a method for characterizing an immunogenic composition comprising purified capsular polysaccharide having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, wherein the method includes the following steps: quantifying the percentage of units of the purified capsular polysaccharide of Formula I; and comparing the percentage of units of Formula I in the purified capsular polysaccharide with a reference standard.

[0013] In some embodiments, this disclosure provides a method for selecting the acetylation level of a purified capsular polysaccharide for use in immunogenic compositions, the method comprising the steps of administering to each of a variety of animals one of a variety of compositions comprising a purified capsular polysaccharide having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, wherein in each of the multiple compositions, R is –C(O)-CH3, which is a unique percentage of the total number of units of Formula I; determining the level of immune response to the purified capsular polysaccharide; comparing the level of immune response with a reference level; and if the determined immune response is higher than the reference level, selecting a purified capsular polysaccharide with a given acetylation level and incorporating it into the immunogenic composition. Brief description of the attached figures

[0014] The teachings described herein will be more fully understood by reading the following description of various illustrative embodiments in conjunction with the accompanying drawings. It should be understood that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the teachings in any way.

[0015] Figure 1 It is obtained by acid hydrolysis in 2M TFA at 120 °C for 2 hours. Streptococcus pneumoniae HPAEC-PAD map of serum type 38 capsular polysaccharide.

[0016] Figure 2 yes Streptococcus pneumoniae ESI-Q-TOF MS spectra of fragments produced by alkaline treatment of serum type 38 capsular polysaccharide.

[0017] Figure 3 Showing natural and reduced Streptococcus pneumoniae GC-MS spectrum of PMMA of serum type 38 capsular polysaccharide.

[0018] Figure 4 A PMMAA derivative corresponding to the reduced Sugp moiety of Spn38 polysaccharide is described.

[0019] Figure 5 MS fragments of Sugp’s PMMA, reconstructed at 23.8 and 24.6 min, are described.

[0020] Figure 6 The natural (trace a) and de-O-acetylated (trace b) sequences are recorded at 600 MHz and 323 K, respectively. pneumococcus bacteria Serum type 38 capsular polysaccharide 1 H NMR spectrum.

[0021] Figure 7 De-O-acetylation recorded at 600 MHz and 323 K Streptococcus pneumoniae Serum type 38 capsular polysaccharide 1 H- 13 C HSQC spectrum. Key repeating unit proton / carbon cross peaks have been labeled.

[0022] Figure 8 De-O-acetylation and reductive de-O-acetylation are described. Streptococcus pneumoniae Partial HSQC NMR results of serum type 38 capsular polysaccharide.

[0023] Figure 9 Natural samples collected at 242 MHz and 323 K Streptococcus pneumoniae Serum type 38 capsular polysaccharide 31 P NMR spectrum.

[0024] Figure 10 This was achieved by selectively irradiating de-O-acetylation recorded at 600 MHz and 323 K. Streptococcus pneumoniae Collected by collecting anomeric signals of serum type 38 capsular polysaccharide. 1 H 1D TOCSY (120 ms mixing time) and 1D NOESY (200 ms mixing time) NMR spectra. Trace a) 1 H reference spectrum; trace b) selective TOCSY H1A; trace c) selective NOESY H1A; trace d) selective TOCSY H1B; trace e) selective NOESY H1B; trace f) selective TOCSY H1C; trace g) selective NOESY H1C; trace h) selective TOCSY H1D and E; trace i) selective NOESY H1D and E.

[0025] Figure 11 De-O-acetylation recorded at 600 MHz and 323 K Streptococcus pneumoniae 2D NOESY spectrum of serum-type 38 capsular polysaccharide. Key repeating unit proton / carbon cross peaks of glycosidic bonds have been labeled and confirmed.

[0026] Figure 12 De-O-acetylation recorded at 600 MHz and 323 K Streptococcus pneumoniae Serum type 38 capsular polysaccharide 1 H- 13 C HMBC spectrum. Key repeating unit proton / carbon cross peaks of glycosidic bonds have been identified and confirmed.

[0027] Figure 13 It is de-O-acetylation Streptococcus pneumoniae A portion of serum type 38 capsular polysaccharide 1 H- 13 C HMBC spectrum.

[0028] Figures 14A-14D Natural records at 600 MHz and 323 K Streptococcus pneumoniae A portion of serum type 38 capsular polysaccharide 1 H -1 H TOCSY ( Figure 14A COSY Figure 14B NOESY Figure 14C ) and HMBC ( Figure 14D ) spectrum. The key repeating unit proton / carbon cross peak at the O-acetylation site of the confirmed α-Galp residue C2 has been identified.

[0029] Figure 15 yes Streptococcus pneumoniae An exemplary structure of a serum-type 38-capsular polysaccharide repeating unit.

[0030] Figure 16 This is a schematic diagram of an exemplary CP1 fusion protein. Such exemplary CP1 fusion proteins include biotin-binding proteins, such as truncated rhizobiamic avidin proteins (e.g., amino acid 45-179 of wild-type rhizobiamic avidin proteins), a first linker (e.g., the GGGGSSS (SEQ ID NO:30) linker), and an SP1500 polypeptide (e.g., a full-length...). Streptococcus pneumoniae The SP1500 peptide contains amino acids 27-278), a second linker (e.g., amino acid sequence AAA), and the SP0785 peptide (e.g., full-length amino acid sequence AAA). Streptococcus pneumoniae The SP0785 peptide contains amino acids 33-399. In some embodiments, the CP1 fusion protein may also include a detectable or purifiable tag (e.g., a His tag). The amino acid sequence AAA may be derived from the Not I site on the pET21 / 24 plasmid or may be synthetic. For the GGGGSSS (SEQ ID NO:30) adapter, the SSS amino acid sequence may be derived from the Sac I site on the pET21 / 24 plasmid, with the GGGG (SEQ ID NO:59) amino acid sequence added to create a flexible adapter with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO:30) adapter may be synthetic.

[0031] Figure 17 This is a schematic diagram of an exemplary SPP2 fusion protein. Such exemplary SPP2 fusion proteins include biotin-binding proteins, such as truncated rhizobiamic avidin proteins (e.g., amino acid 45-179 of wild-type rhizobiamic avidin protein, referred to as Rhavi), a first linker (e.g., the GGGGSSS (SEQ ID NO:30) linker), and a pneumococcal hemolysin (Ply) polypeptide (e.g., full-length). Streptococcus pneumoniae The Ply polypeptide comprises amino acids 2-470, which includes the mutants G294P, D385N, C428G, and W433F, referred to as PdT(G294P)), a second linker (e.g., the GGGGSSS (SEQ ID NO:30) linker), and the SP0435 polypeptide (e.g., the full-length). lung Streptococcus inflammatoryaeThe SP0435 polypeptide contains amino acids 62-185. In some embodiments, the SPP2 fusion protein may also include a detection or purification tag (e.g., a His tag). For the GGGGSSS (SEQ ID NO: 30) adapter, the SSS amino acid sequence may be derived from the Sac I site on the pET21 / 24 plasmid, with the GGGG (SEQ ID NO: 59) amino acid sequence added to create a flexible adapter with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO: 30) adapter may be synthetic.

[0032] Figure 18 This is a schematic diagram of a plasmid construct consisting of a pET-24a(+) vector containing a sequence encoding an exemplary SPP2 fusion protein.

[0033] Figure 19 and 20 It is an exemplary upstream of fusion proteins (e.g., CP1 and SPP2) as described herein. Figure 19 ) and downstream ( Figure 20 The flowchart illustrates the manufacturing process. An exemplary SPP2 peptide is a fusion protein comprising a truncated rhizobiacin [amino acids 45-179 of the full-length rhizobiacin protein], a first linker (e.g., the GGGGSSS linker), a pneumococcal hemolysin (Ply) peptide, a second linker (e.g., the GGGGSSS linker), and an SP0435 peptide. An exemplary CP1 peptide is a fusion protein comprising a truncated rhizobiacin [amino acids 45-179 of the full-length rhizobiacin protein], a first linker (e.g., the GGGGSSS linker), an SP1500 peptide, a second linker (e.g., amino acid sequence AAA), and an SP0785 peptide. SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFF: Tangential flow filtration; DS: Active pharmaceutical ingredient.

[0034] Figure 21 It displays serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. Streptococcus pneumoniae A table of exemplary structures of antigenic polysaccharides.

[0035] Figure 22This is a schematic diagram of an exemplary multiplex antigen presentation system (MAPS). In the illustrated exemplary embodiment, the MAPS immunogenic complex comprises one or more polypeptide antigens fused to a biotin-binding protein, rhizobinacin, or its biotin-binding domain or biotin-binding fragment, and a biotinylated antigenic polysaccharide. In this figure, each MAPS complex is formed between one or more fusion proteins and a biotinylated polysaccharide by non-covalent binding of a truncated rhizobinacin to biotin.

[0036] Figure 23 This is a flowchart describing an exemplary manufacturing process for the MAPS immunogenic complex, including the biotinylation of the antigenic polysaccharide and its assembly with a fusion protein (e.g., CP1 or SPP2) as described herein. MAPS: Multiple Antigen Presentation System.

[0037] Figure 24 This is a flowchart describing an exemplary manufacturing process for a MAPS vaccine. For example, such an exemplary process can be used to manufacture a MAPS34 vaccine comprising MAPS immunogenic complexes from 34 different... Streptococcus pneumoniae Serum-type capsular polysaccharide. DMAP: 4-dimethylaminopyridine; MAPS: multiplex antigen presentation system; PS: polysaccharide. †Testing in progress.

[0038] Figure 25 The results of nephrological tests using a mixed serum from 10 rabbits inoculated with PS38-CRM197 are described.

[0039] Figure 26 The ELISA results of rabbit serum prepared by immunization against PS38-CRM197 are described.

[0040] Figure 27 ELISA results of rabbit serum following vaccination with a multivalent MAPS vaccine containing PS-38 in combination with CP1 or SPP2 are described. Serum was collected from male NZW rabbits (n=20) two weeks before the first dose (P0), before the second dose (P1), and after the second dose (P2). The concentration of anti-ST-38 specific IgG was determined by comparing the samples with a reference standard of known concentration. Horizontal bars represent the geometric mean concentration for each group, and error bars represent 95% confidence intervals. Detailed Implementation

[0041] Among other things, this disclosure provides Streptococcus pneumoniae Purified capsular polysaccharide of serotype 38, and an immunogenic composition comprising said purified capsular polysaccharide.

[0042] definition

[0043] The compounds disclosed herein include those generally described above, and are further described by the classes, subclasses and species disclosed herein. Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements have been identified according to the periodic table (CAS edition) and the Handbook of Chemistry and Physics (75th edition). Furthermore, the general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University ScienceBooks, Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5th edition, edited by Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0044] Unless otherwise stated, the structures described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the invention include those using deuterium or tritium instead of hydrogen, or those using... 13 C or 14 All carbon substitutions for C enrichment are within the scope of this disclosure.

[0045] About or Approximately As used herein, the terms "about" or "approximately" apply to one or more values ​​of interest, referring to values ​​similar to the reference value. Generally, those skilled in the art will understand the extent of variation covered by "about" or "approximately" in this context. For example, in some embodiments, the terms "about" or "approximately" may cover a range of values ​​within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the mentioned value.

[0046] Application:As used herein, the terms "administering" or "administration" generally refer to administering a composition to a subject to achieve delivery as a composition or a drug contained in a composition to a target site or therapeutic site. Those skilled in the art will recognize that a variety of routes can be used to administer the composition to a subject (e.g., a person) where appropriate. For example, in some embodiments, administration may be via the eye, mouth, parenteral, topical, etc. In some specific embodiments, administration may be via the bronchus (e.g., via bronchial infusion), buccal, percutaneous (which may be or include, for example, one or more of the following: topical to the dermis, intradermal, intradermal, transdermal, etc.), intestinal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., via tracheal infusion), vaginal, vitreous, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some specific embodiments, administration may be intravenous. In some specific embodiments, administration may be subcutaneous. In some embodiments, administration may involve a single dose only. In some embodiments, administration may involve applying a fixed number of doses. In some embodiments, administration may involve administration as intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses separated by a common time period). In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected time period. In some embodiments, administration may include a primary and booster regimen. A primary and booster regimen may include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine), followed by administration of a second or subsequent dose of the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) after a period of time. In the case of an immunogenic composition, a primary and booster regimen may result in an increased immune response in the subject.

[0047] Agent:As used herein, the term "pharmaceutical" can be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Where appropriate, as will be apparent from the context to those skilled in the art, the term can be used to refer to an entity that is or comprises a cell or organism or a fraction, extract, or component thereof. Or, additionally, as will be apparent from the context, the term can be used to refer to a natural product because it is found in and / or obtained from nature. In some instances, further as will be apparent from the context, the term can be used to refer to one or more man-made entities because they are designed, engineered, and / or produced by human intervention and / or are not found in nature. In some embodiments, pharmaceuticals can be utilized in isolated or pure forms; in some embodiments, pharmaceuticals can be utilized in crude substance form. In some embodiments, potential pharmaceuticals can be provided as collections or libraries, for example, they can be screened to identify or characterize the active agents therein. In some instances, the term "pharmaceutical" can refer to a compound or entity that is or comprises a polymer; in some instances, the term can refer to a compound or entity comprising one or more polymeric moieties. In some embodiments, the term "pharmaceutical" may refer to a compound or entity that is not a polymer and / or substantially free of any polymer and / or substantially free of one or more specific polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or substantially free of any polymeric moieties.

[0048] Amino acids:As used herein, the term "amino acid" refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, the amino acid has the general formula H₂N–C(H)(R)–COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is prepared synthetically or obtained from a natural source. In some embodiments, amino acids including carboxyl-terminal and / or amino-terminal amino acids in a polypeptide may contain structural modifications compared to the general formula above. For example, in some embodiments, the amino acid may be modified by methylation, amidation, acetylation, PEGylation, glycosylation, phosphorylation, and / or substitution (e.g., substitution of amino, carboxylic acid, one or more protons and / or hydroxyl groups) compared to the general formula. In some embodiments, such modification may, for example, alter the cycling half-life of a polypeptide containing modified amino acids compared to a polypeptide containing otherwise identical unmodified amino acids. In some embodiments, such modification does not significantly alter the relevant activity of a polypeptide containing modified amino acids compared to a polypeptide containing otherwise identical unmodified amino acids. As will be apparent from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, the term "amino acid" may be used to refer to an amino acid residue of a polypeptide.

[0049] antigen:As used herein, the term "antigen" refers to (i) an agent that induces an immune response; and / or (ii) an agent that binds to a T-cell receptor (e.g., when presented via an MHC molecule) or to an antibody. In some embodiments, an antigen induces a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen induces a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen induces both humoral and cellular responses. In some embodiments, an antigen binds to an antibody and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or includes any chemical entity such as small molecules, nucleic acids, peptides, carbohydrates, lipids, polymers (in some embodiments, any chemical entity other than biopolymers (e.g., other than nucleic acid or amino acid polymers)). In some embodiments, an antigen is or comprises a peptide. In some embodiments, an antigen is or comprises a polysaccharide. Those skilled in the art will understand that, generally, antigens can be provided in isolated or pure form, or alternatively, in crude material form (e.g., together with other materials, such as extracts of cell extracts or other relatively crude formulations containing the antigen). In some embodiments, the antigen utilized according to this disclosure is provided in crude form. In some embodiments, the antigen is a recombinant antigen. In some embodiments, the antigen is a polypeptide or polysaccharide that, upon administration to a subject, induces a specific and / or clinically relevant immune response against the polypeptide or polysaccharide. In some embodiments, the antigen is selected to induce a specific and / or clinically relevant immune response against the polypeptide or polysaccharide. Those skilled in the art will recognize that the antigen may be a full-length polypeptide antigen, or a portion thereof comprising one or more B-cell epitopes and / or one or more T-cell epitopes.

[0050] Associated with: As used herein, two entities are “associated” with each other if the presence, level, and / or form of one entity is related to the presence, level, and / or form of another entity. In some embodiments, two or more entities are physically “associated” with each other if they interact directly or indirectly to bring them physically close to each other and / or keep them physically close. In some embodiments, two or more entities that are physically associated with each other are covalently connected. In some embodiments, two or more entities that are physically associated with each other are non-covalently connected, but non-covalent association occurs, for example, by means of affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0051] Biotin-binding portion: As used herein, the term "biotin-binding moiety" refers to a biotin-binding protein, or a biotin-binding fragment, or a biotin-binding domain.

[0052] Combination: It should be understood that, as used herein, the term “binding” generally refers to non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or parts; indirect binding involves physical interaction by means of physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated in any of a variety of situations – including in cases where the interacting entities or parts are isolated or in cases of more complex systems (e.g., when covalently or otherwise associated with a carrier entity and / or within a biological system or cell).

[0053] Biological samples: As used herein, the term "biological sample" generally refers to a sample obtained from or derived from a biological source of interest as described herein (e.g., a tissue or organism or cell culture). In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or comprises biological tissue or fluid. In some embodiments, the biological sample may be or comprises bone marrow; blood; blood cells; ascites; tissue or fine-needle aspiration biopsy samples; body fluids containing cells; cell-free nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; lavage or flushing solutions, such as catheter lavage or bronchoalveolar lavage fluid; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions and / or excretions; and / or cells therein, etc. In some embodiments, the biological sample is or comprises cells from an individual. In some embodiments, the obtained cells are or include cells from the individual from whom the sample was obtained. In some embodiments, the sample is a “raw sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, the raw biological sample is obtained by methods selected from: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term “sample” refers to a preparation obtained by processing (e.g., by removing one or more components from the raw sample and / or adding one or more reagents thereto) the raw sample. For example, using semi-permeable membrane filtration. Such a “processed sample” can include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the raw sample to techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components.

[0054] Carrier:As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or transporter used to administer a composition. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

[0055] Carrier proteins: As used herein, the term "carrier protein" refers to a protein or peptide that is conjugated, complexed, or otherwise associated with a hapten (e.g., a small peptide or lipid) or a less immunogenic antigen (e.g., a polysaccharide), and induces or improves an immune response against such conjugated, complexed, or otherwise associated hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g., a polysaccharide). In some embodiments, such an immune response is or comprises a response against a hapten or less immunogenic antigen conjugated, complexed, or otherwise associated with such a carrier protein. In some embodiments, such an immune response is or comprises a response against both the carrier protein and the hapten or less immunogenic antigen conjugated, complexed, or otherwise associated with such a carrier protein. In some embodiments, no significant immune response occurs against the carrier protein itself. In some embodiments, an immune response against the carrier protein can be detected; in some embodiments, an immune response against such a carrier protein is strong. In some embodiments, the carrier protein is conjugated, complexed, or otherwise associated with one or more other molecules.

[0056] colonization: As used herein, the term “colonization” generally refers to the ability of microorganisms to grow at a target site or surface. For example, the term “colonization” refers to the ability of microorganisms (e.g., bacteria) to grow at anatomical sites of a host (e.g., mucous membranes, gastrointestinal tract, sites of injury, organs, etc.).

[0057] Combination therapy:As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents or methods). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., a first regimen with all "doses" administered before any dose of a second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve administering one or more agents or regimens to a subject receiving other agents or regimens in combination form. For clarity, combination therapy does not require individual agents to be administered together as a single composition (or even simultaneously), but in some embodiments, two or more agents or their active portions may be administered together as a combination composition or even as a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0058] Comparable: As used herein, the term "comparable" refers to two or more sets of reagents, entities, situations, conditions, etc., that may differ from one another but are similar enough to allow for comparison between them, and thus, those skilled in the art will understand, that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, a set of comparable conditions, environments, individuals, or groups is characterized by having several substantially identical features, and one or a few features that differ. Those skilled in the art will understand in context that, in any given situation, two or more such sets of reagents, entities, situations, conditions, etc., are considered to be of the degree of identity required to be comparable. For example, those skilled in the art will understand that a set of situations, individuals, or groups is comparable to one another when characterized by a sufficient number and type of substantially identical features to guarantee a reasonable conclusion that differences in results or observed phenomena obtained under different situations, individuals, groups, or conditions are caused by changes in the features that indicate these changes.

[0059] Composition: Those skilled in the art will understand that the term "composition" can be used to refer to a discrete physical entity comprising one or more specific components. Generally, unless otherwise stated, a composition can be in any form, such as a gas, gel, liquid, solid, etc.

[0060] Domain:As used herein, the term "domain" refers to a segment or portion of an entity. In some embodiments, a "domain" is associated with a specific structural and / or functional feature of an entity such that it substantially or completely retains that specific structural and / or functional feature when physically separated from the remainder of its parent entity. Alternatively or additionally, a domain may be or comprise a portion of an entity that substantially retains and / or provides on the receptor entity one or more structural and / or functional features that characterize it in the parent entity when separated from the (parent) entity and attached to a different (receptor) entity. In some embodiments, a domain is a segment or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a segment of a polypeptide; in some such embodiments, the domain is characterized by specific structural elements (e.g., a specific amino acid sequence or sequence motif, α-helix feature, β-sheet feature, coil-coil feature, random coil feature, etc.) and / or specific functional features (e.g., binding activity, enzymatic activity, folding activity, signal transduction activity, etc.).

[0061] Dosage form or unit dosage form: Those skilled in the art will understand that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) administered to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, this amount is an amount (or a whole portion thereof) suitable for administering a unit dose according to a dosing regimen determined to be relevant to the desired or beneficial outcome when administered to the relevant population (i.e., using a therapeutic dosing regimen).

[0062] Dosing regimen or treatment plan:Those skilled in the art will understand that the terms "dosing regimen" and "treatment regimen" can be used to refer to a set of unit doses (usually more than one) administered individually to a subject at regular intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each time-spaced from the other doses. In some embodiments, the doses are spaced apart from each other by the same length of time; in some embodiments, a dosing regimen includes multiple doses and at least two different time intervals separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dose amount. In some embodiments, the different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount different from the first dose amount. In some embodiments, a dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount identical to the first dose amount. In some embodiments, a dosing regimen is associated with a desired or beneficial outcome when administered to a relevant population (i.e., a therapeutic dosing regimen).

[0063] excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or promote a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.

[0064] Excerpt:As described herein, a "fragment" of a material or entity has discrete parts that comprise the whole, but lacks the structure of one or more parts found in the whole. In some embodiments, the fragment is composed of such discrete parts. In some embodiments, the fragment includes discrete parts of the whole that share one or more functional features found in the whole. In some embodiments, the fragment is composed of such discrete parts. In some embodiments, the fragment consists of or includes the following: characteristic structural elements or portions found in the whole. In some embodiments, fragments of polymers, such as polypeptides or polysaccharides, comprise or consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) found in the whole polymer. In some embodiments, the polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of monomer units (e.g., residues) found in the whole polymer. In some embodiments, the whole material or entity may be referred to as the “parent” of the whole.

[0065] Homology:As used herein, the term "homology" refers to the overall correlation between aggregate molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, aggregate molecules are considered "homological" if their sequences have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. In some embodiments, polymeric molecules are considered "homologous" to each other if their sequences have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity (e.g., containing residues with relevant chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are often similarly classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains. The substitution of one amino acid for another of the same type can often be considered a "homologous" substitution.

[0066] Identity:As used herein, the term "identity" refers to the overall relevance between aggregate molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, aggregate molecules are considered "substantially identical" to each other if the aggregate molecules have an identity of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. For example, the calculation of the identity percentage of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., vacancies can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some implementations, the length of the sequence being compared for comparative purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence, the molecule is considered identical at that position. The percentage of identity between two sequences varies with the number of shared congruent positions, taking into account the number of vacancies that need to be introduced for optimal alignment and the length of each vacancy. Sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the algorithm of Meyers and Miller, 1989, incorporated into the ALIGN program (version 2.0), can be used to determine the percentage of identity between two nucleotide sequences. In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program employ a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0067] Improve, increase, inhibit, or reduce:As used herein, the terms “improvement,” “increase,” “inhibition,” “reduction,” or their grammatical equivalents indicate a value relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or include a measurement in a specific system (e.g., in a single subject) that is otherwise equivalent in the absence (e.g., before and / or after) of a specific agent or treatment, or in the presence of an appropriate equivalent reference agent. In some embodiments, an appropriate reference measurement may be or include a measurement in an equivalent system that is known or expected to respond in a specific manner in the presence of a relevant agent or treatment.

[0068] Effective immunization dose or effective immunization amount: As used herein, "immunogenically effective amount" or "immunogenic dose" refers to an amount of antigenic or immunogenic substance, such as an antigen, immunogen, immunogenic agent, immunogenic composition, vaccine composition, or pharmaceutical composition, which, when administered to a subject in a single dose or as part of a series of doses, is sufficient to enhance the subject's own immune response to subsequent exposure to a pathogen. In some embodiments, the pathogen is... Streptococcus pneumoniae In some implementations, the immune response targets Streptococcus pneumoniae One or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae Two or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae Nine or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae Thirteen or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae Fifteen or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae There are twenty-three or more different serotypes. In some implementations, the immune response targets... Streptococcus pneumoniae There are twenty-four or more different serotypes. The immunologically effective dose can vary based on the subject to be treated, the species of the subject, the degree of immune response to be induced, etc. In some embodiments, the immunologically effective dose is sufficient to treat or protect a subject who has a disease or is at risk of having a disease. In some embodiments, the immunologically effective dose refers to a non-toxic but sufficient amount, which can be an amount used to treat, attenuate, or prevent infection and / or disease (e.g., bacterial infection, pneumococcal infection, bacterial colonization, pneumococcal colonization, complications associated with bacterial infection, complications associated with pneumococcal infection, etc.) in any subject. In some embodiments, the immunologically effective dose is sufficient to induce a protective immune response after administration to the subject.

[0069] Immune protective response or protective response:As used herein, "immunoprotective response" or "protective response" refers to an immune response that mediates an antigen- or immunogen-induced immunological memory. In some embodiments, an immune protective response is induced by administering to a subject a substance such as an antigen, immunogen, immunogenic agent, immunogenic composition, vaccine composition, or pharmaceutical composition. In some embodiments, immune protection involves one or more of the following: active immune surveillance, a more rapid and effective post-activated immune response compared to a response observed in unimmunized subjects, effective clearance of the activator or pathogen, followed by rapid resolution of inflammation. In some embodiments, the immune protective response is an adaptive immune response. In some embodiments, the immune protective response is sufficient to protect an immunized subject from toxogenic infections caused by one or more specific pathogens targeted by the vaccine (e.g., Streptococcus pneumoniae Infect).

[0070] Immunization: As used herein, “immunity” or its grammatical equivalent refers to the process of inducing an immune response (“active immunization”) in a subject against an infectious organism or agent, or alternatively, the process of providing a subject with components of the immune system against an infectious organism or agent (“passive immunization”). In some embodiments, immunization involves administering to a subject one or more antigens, immunogens, immunogenic agents, immunogenic compositions, vaccine compositions, immune molecules (such as antibodies), immune serum, immune cells (such as T cells or B cells), or pharmaceutical compositions. In some embodiments, immunization is performed by administering to a subject an immunologically effective amount of a substance such as an antigen, immunogen, immunogenic agent, immunogenic composition, vaccine composition, immune molecule (such as antibody), immune serum, immune cells (such as T cells or B cells), or pharmaceutical composition. In some embodiments, immunization elicits a protective immune response in a subject. In some embodiments, active immunization is performed by administering to a subject an antigenic or immunogenic substance such as an antigen, immunogen, immunogenic agent, immunogenic composition, vaccine composition, or pharmaceutical composition. In some implementations, passive immunization is performed by administering immune system components, such as immune molecules (e.g., antibodies), immune serum, or immune cells (e.g., T cells or B cells), to the subject.

[0071] Isolated:As used herein, the term “separated” or its grammatical equivalent means a substance and / or entity that has (1) been separated from at least some of the components with which it was initially associated (whether in nature and / or in an experimental setting), and / or (2) been artificially designed, produced, prepared and / or manufactured. A separated substance and / or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which it was initially associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is considered "pure" if it is substantially free of other components. In some embodiments, as those skilled in the art will understand, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance is calculated without excluding these carriers or excipients. In some embodiments, for the sake of example only, biopolymers such as polypeptides or polysaccharides that occur in nature are considered "isolated" if: a) they are not associated with some or all of the components that accompany them in their native state in nature due to their derivation or origin; b) they are substantially free of other polypeptides or nucleic acids of the same kind as those that produce them in nature; or c) they are expressed or otherwise associated with components from cells or other expression systems that do not have the kind that produce them in nature. Thus, for example, in some embodiments, polypeptides or polysaccharides that are chemically synthesized or synthesized in a cellular system different from the cellular system that produces them in nature are considered "isolated" polypeptides or polysaccharides. Alternatively or additionally, in some embodiments, polypeptides or polysaccharides that have undergone one or more purification techniques may be considered "isolated" polypeptides or polysaccharides to the extent that they have been isolated from a) those associated with them in nature; and / or b) other components associated with them at the time of their initial production.

[0072] Connector:As used herein, the term "connector" refers to an entity that links two or more elements to form a multi-element pharmaceutical agent. For example, those skilled in the art will understand that peptides whose structure includes two or more functional or tissue domains often include a segment of amino acids between such domains that connect them to each other. In some embodiments, peptides containing a connector element have an overall structure of the general form S1-L-S2, where S1 and S2 can be the same or different and represent two domains associated with each other by the connector (L). In some embodiments, the length of the peptide connector is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized by its tendency to provide flexibility to the peptide rather than employing a rigid three-dimensional structure. A variety of different linker elements that can be suitably used when engineering peptides (e.g., fusion peptides) are known in the art (Holliger et al., 1993; Poljak, 1994).

[0073] mutation: As used herein, the term "mutation" refers to a difference in the sequence of a nucleic acid or polypeptide compared to a reference sequence. Exemplary types of mutations include, but are not limited to, insertions, deletions, and substitutions. For the avoidance of doubt, the term "mutation" should be understood by those skilled in the art to include differences compared to a reference sequence and does not necessarily refer to or imply a change occurring within any particular target sequence.

[0074] Non-inferior:As used herein, the term "non-inferiority" in the context of evaluating a test pharmaceutical composition means that the test pharmaceutical composition (e.g., in terms of the immunogenicity and / or functional antibody titer generated by the test composition) is at least as effective as the reference composition. In some embodiments, non-inferiority is demonstrated when the lower limit of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of the test pharmaceutical composition to the reference composition is at least greater than or equal to 0.4 or higher, including, for example, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 0.95, at least 0.98, at least 1.0 or higher. For example, in some embodiments, the immunogenic composition (e.g., vaccine) is not inferior to the reference vaccine (e.g., PCV13, PCV20, or PPSV23) when the lower limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to a reference vaccine is at least greater than or equal to 0.4, including, for example, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 0.95, at least 0.98, at least 1.0 or higher. In some embodiments, the immunogenic composition (e.g., vaccine) is not inferior to the reference vaccine (e.g., PCV13, PCV20, or PPSV23) when the lower limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to a reference vaccine is at least greater than or equal to 0.95, including, for example, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 1.0, at least 1.1, at least 1.3, at least 1.5, or higher. In some embodiments, the immunogenic composition (e.g., vaccine) described herein is not inferior to the reference vaccine (e.g., PCV13, PCV20, or PPSV23) when the seroconversion rate or the percentage of vaccine recipients with an immune response is higher than a predetermined threshold, for example, when the lower limit of the 95% confidence interval of the difference between the percentage of subjects seroconverted after immunization with the immunogenic composition (e.g., vaccine) described herein and immunization with a reference vaccine (e.g., PCV13, PCV20, or PPSV23) is greater than -0.10.

[0075] extraintestinal:As used herein, the phrase “parenteral administration / administered parenterally” has the meaning as understood in the art, referring to a mode of administration other than enteral and local administration, usually administered by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, and intrasternal injections and infusions.

[0076] Patient or subject: As used herein, the terms "patient" or "subject" refer to any organism to which the provided composition is applied or may be applied, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient or subject suffers from or is susceptible to one or more diseases or conditions. In some embodiments, the patient or subject exhibits symptoms of one or more diseases or conditions. In some embodiments, the patient or subject has been diagnosed with one or more diseases or conditions. In some embodiments, the patient or subject is receiving or has received a therapy for the diagnosis and / or treatment of a disease, disease, or condition.

[0077] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a treatment or dosing regimen at a unit dose suitable for administration, which shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as oral enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual, and systemic absorption), pills, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension or a sustained-release formulation; topical administration, such as as a cream, ointment, or controlled-release patch, or a spray for application to the skin, lungs, or mouth; intravaginal or rectal administration, such as as a cervical cap, cream, or foam; sublingual administration; ocular administration; transdermal administration; or nasal administration, pulmonary administration, and administration to other mucosal surfaces.

[0078] Pharmaceutically acceptable:As used herein, the phrase “pharmaceutically acceptable” means, to a reasonable extent of medical judgment, that a compound, material, composition, and / or dosage form is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0079] Prevention or prevention: As used herein, the terms “prevent” or “prevention”, when used in conjunction with the occurrence of a disease, condition, and / or illness, refer to reducing the risk of a patient’s disease, condition, and / or illness and / or delaying the onset of one or more features or symptoms of a disease, condition, or illness. Prevention can be considered complete when the onset of a disease, condition, or illness has been delayed for a predetermined period of time.

[0080] Polysaccharides: As used herein, the term "polysaccharide" refers to a polymeric carbohydrate molecule consisting of long chains of monosaccharide units linked together by glycosides, phosphodiester bonds, or other bonds, and which, upon hydrolysis, yields the constituent monosaccharides or oligosaccharides. The structural range of polysaccharides is from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen; structural polysaccharides such as cellulose and chitin, and microbial polysaccharides; and antigenic polysaccharides found in microorganisms, including but not limited to capsular polysaccharides (CPS), O-polysaccharides (OPS), core O-polysaccharides (COPS), and lipopolysaccharides (LPS).

[0081] polypeptide:As used herein, the term "peptide" generally has the meaning, as is generally understood in the art, of a polymer of at least three amino acids linked together by peptide bonds. Those skilled in the art will understand that the term "peptide" is intended to be sufficiently general to encompass not only peptides having the complete sequences listed herein, but also peptides representing functional fragments of these complete peptides (i.e., fragments that retain at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally allow for some substitutions without destroying activity. Therefore, as used herein, the relevant term "peptide" encompasses any peptide that retains activity and shares at least about 30-40% (often greater than about 50%, 60%, 70%, or 80%) of overall sequence identity with another peptide of the same class, and further generally includes any peptide that typically encompasses at least 3-4 amino acids, and often up to 20 or more, having at least a significantly higher identity (often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions). Peptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0082] Prevention: As used herein in connection with diseases, symptoms, and / or medical conditions, the terms "prevent" or "prevention" refer to reducing the risk of developing a disease, symptom, and / or condition, and / or delaying its onset, and / or reducing the frequency and / or severity of one or more features or symptoms of a particular disease, symptom, or condition. In some embodiments, prevention is evaluated on a population basis such that the agent is considered to have "prevented" a particular disease, symptom, or condition if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of the disease, symptom, or condition is observed in a population susceptible to the disease, symptom, or condition. In some embodiments, prevention may be considered complete when the onset of a disease, symptom, or condition has been delayed for a predetermined period of time.

[0083] protein:As used herein, the term "protein" encompasses polypeptides. Proteins may include portions other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those skilled in the art will understand that a "protein" can be a complete polypeptide chain produced by cells (with or without a signal sequence), or it can be a characteristic portion thereof. Those skilled in the art will understand that proteins may sometimes include more than one polypeptide chain linked by one or more disulfide bonds or otherwise associated. Polypeptides may contain l-amino acids, d-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides with a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, its biologically active portion, and / or a characteristic portion thereof.

[0084] Reorganization: As used herein, the term "recombinant" is intended to mean a polypeptide that is designed, engineered, prepared, expressed, generated, manufactured, and / or isolated by recombinant means, such as polypeptides, including polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant combined human polypeptide library; polypeptides isolated from transgenic or otherwise manipulated genomic components of animals (e.g., mice, rabbits, sheep, fish, etc.) that express one or more genes or genes encoding and / or guiding the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or polypeptides prepared, expressed, generated, or isolated by any other means involving splicing selected nucleic acid sequence elements or linking selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids encoding and / or guiding the expression of a polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed by computer simulation. In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, such as natural or synthetic sources from a strain of a target source organism (e.g., human, mouse, etc.).

[0085] refer to:As used herein, the term "reference" describes a standard or control relative to the comparison performed. For example, in some embodiments, a target agent, animal, subject, population, sample, sequence, or value is compared with a reference or control agent, animal, subject, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or measured substantially concurrently with the target test or assay. In some embodiments, the reference or control is optionally a historical reference or control performed in a tangible medium. Generally, as those skilled in the art will understand, the reference or control is measured or characterized under conditions or circumstances equivalent to those being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify reliance on and / or comparison with a particular probability reference or control.

[0086] answer: As used herein, a “response” to treatment may refer to any beneficial change in a subject’s condition caused by or related to treatment. Such a change may include stabilization of the condition (e.g., prevention of exacerbations that would occur in the absence of treatment), improvement of symptoms, and / or improved prospects for cure. A subject’s response may be measured according to a wide range of criteria, including clinical and objective criteria. Techniques used to assess response include, but are not limited to, clinical examination, positron emission tomography (PET), chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, the presence or level of biomarkers in samples obtained from the subject, cytology, and / or histology. Accurate response criteria may be selected in any appropriate manner, provided that when comparing subject groups, the groups being compared are assessed based on the same or equivalent criteria used to measure response rates. Those skilled in the art will be able to select appropriate criteria.

[0087] risk: As will be understood from the context, the “risk” of a disease, condition, and / or illness refers to the likelihood that a particular subject will develop a disease, condition, and / or illness. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%. In some embodiments, risk is expressed as risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known disease, condition, illness, and / or event risk. In some embodiments, the reference sample or reference sample group is drawn from subjects comparable to the particular subject. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0088] Serotype:As used herein, the term "serotype," also known as serovar, refers to variations within a bacterial or viral species or in the immune cells of different subjects. These microorganisms, viruses, or cells are grouped together based on their cell surface antigens, allowing for epidemiological classification of organisms down to the subspecies level. Groups of serovars with common antigens may be referred to as serogroups or sometimes as seroclusters.

[0089] Species: As used herein, the term "species" refers to different immunogenic compositions comprising (i) a biotinylated polysaccharide antigen, and (ii) a biotinylated binding moiety and one or more Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae A fusion protein containing a polypeptide antigen. In some embodiments, different species may use (i) a biotinylated polysaccharide antigen and (ii) a protein containing a biotin-binding moiety and one or more Streptococcus pneumoniae (Streptococcus pneumoniae) antigens. Streptococcus pneumoniae The differences lie in one or more of the fusion proteins of the polypeptide antigen.

[0090] treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method that partially or completely relieves, improves, reduces, inhibits, prevents, delays the onset of one or more symptoms or features of a disease, disorder, and / or condition, reduces their severity, and / or decreases their incidence. Treatment may be administered to subjects who do not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who only exhibit early signs of a disease, disorder, and / or condition, for example, to reduce the risk of pathological development associated with the disease, disorder, and / or condition.

[0091] Vaccination: As used herein, the term "vaccination" refers, for example, the administration of a composition intended to generate an immune response to a pathogen. For the purposes of this disclosure, vaccination may be administered before, during, and / or after exposure to a pathogen, and in some embodiments shortly before, during, and / or after exposure to a pathogen. In some embodiments, vaccination comprises multiple administrations of a vaccination composition at appropriate time intervals. In some embodiments, vaccination induces immunity.

[0092] Variants:As used herein in the context of molecules such as nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, for example, in the presence or absence of one or more chemical motifs or at a certain level. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity between the particular molecule and the reference molecule. As those skilled in the art will appreciate, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements with a reference molecule but differs from it in at least one respect. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to one or more differences in the amino acid or nucleotide sequence and / or one or more differences in chemical motifs (e.g., carbohydrates, lipids, phosphate groups), which are covalent components of the polypeptide or nucleic acid (e.g., linked to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities with the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, if the target polypeptide or nucleic acid has an amino acid or nucleotide sequence that is identical to the reference polypeptide or nucleic acid but has a few sequence variations at specific positions, the polypeptide or nucleic acid is considered a "variant" of the reference polypeptide or nucleic acid. Typically, compared to the reference, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residues in the variant are substituted, inserted, or deleted. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. Typically, the variant polypeptide or nucleic acid contains a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in specific biological activities) relative to the reference.In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, it does not contain any additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically fewer than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically fewer than about 5, about 4, about 3, or about 2 modifications (e.g., substitution, addition, or deletion) in the N-terminal portion. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically fewer than about 5, about 4, about 3, or about 2 modifications (e.g., substitution, addition, or deletion) in the C-terminal portion. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

[0093] “ "Refers to the connection point between two atoms."

[0094] Immunogenic drugs Purified capsular polysaccharide of serotype 38

[0095] Among other things, this disclosure provides for features not previously characterized. Streptococcus pneumoniae This document outlines research insights and technical solutions related to the discovery and characterization of serotype 38. In some embodiments, the research insights and technical solutions provided herein are used to provide immunogenic compositions (e.g., vaccines) with enhanced immunogenicity to offer protection against certain diseases, conditions, and illnesses, such as those targeting specific diseases. Streptococcus pneumoniae Those related to serotype 38.

[0096] In some embodiments, this disclosure provides a composition comprising purified capsular polysaccharides having repeating units of Formula I: I Each R is independently selected from H and –C(O)-CH3. In some embodiments, R is H. In some embodiments, R is –C(O)-CH3. In some embodiments, the purified capsular polysaccharide comprises between 1 and about 2000 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 1000 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 750 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 500 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 250 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 100 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or about 200 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 4 and about 500 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 4 and about 400 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises between about 1 and about 10 units of Formula I. In some embodiments, the purified capsular polysaccharide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9 or about 10 units of Formula I.

[0097] In some embodiments, approximately 60% of the repeating units in the purified capsular polysaccharide of Formula I are in α-Gal f The residue at C2 is O-acetylated (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains greater than or equal to 60% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein comprises 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% in α-Gal fThe C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains about 60 to about 70% repeating units, wherein R in Formula I is –C(O)-CH3. In some embodiments, the purified capsular polysaccharide described herein contains 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70% repeating units, wherein R in Formula I is –C(O)-CH3.

[0098] In some embodiments, the purified capsular polysaccharide described herein contains between 0% and 100% α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein contains about 0% to about 75% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3).

[0099] In some embodiments, the purified capsular polysaccharide described herein contains less than 60% of α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3). In some embodiments, the purified capsular polysaccharide described herein comprises 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0% in α-Gal f The C2 position of the residue is an O-acetylated repeating unit (i.e., R in Formula I is –C(O)-CH3).

[0100] In some embodiments, the purified capsular polysaccharide of Formula I is biotinylated (i.e., "biotinylated purified capsular polysaccharide").

[0101] In some embodiments, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 300 kDa or higher. In some embodiments, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 5000 Daltons or lower. In some embodiments, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 600 kDa to about 4000 kDa. In some embodiments, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 3675 Daltons to about 4000 kDa. In one embodiment, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 600 kDa. In another embodiment, the purified capsular polysaccharide of Formula I has a weight-average molecular weight (Mw) of about 3550 kDa.

[0102] In some embodiments, this disclosure provides a purified capsular polysaccharide of Formula I, which has been activated by or has been activated by a chemical agent, such as an oxidizing agent or a reducing agent. As described herein, a capsular polysaccharide is “activated” when it has been chemically modified to enable conjugation with a linker or carrier protein. For example, in some embodiments, the purified capsular polysaccharide of Formula I is activated by a chemical agent (e.g., an oxidizing agent or a reducing agent) to generate a reactive group for conjugation with a suitable conjugate (e.g., a linker or carrier protein). In some embodiments, the reactive group is an aldehyde group (-C(O)H). In some embodiments, the reactive group is a cyanate group (-OCN). In some embodiments, activation occurs in β-Gal f At C2, C3, C5, or C6 of the residue (i.e., at the position designated as R in Formula I above). In some embodiments, activation is achieved by oxidative cleavage of the monosaccharide exchange, thereby producing a moiety having one or more aldehyde, cyanate, or amide groups.

[0103] In some embodiments, this disclosure provides an activated capsular polysaccharide comprising one or more units of Formula I. In some embodiments, this disclosure provides an activated serum-type 38 capsular polysaccharide. In some embodiments, an activated serum-type 38 capsular polysaccharide comprises a reactive hydrogen ester group or an aldehyde group.

[0104] In some embodiments, this disclosure provides a purified capsular polysaccharide comprising one or more units of Formula I, wherein the purified capsular polysaccharide is conjugated with a carrier protein or biotin.

[0105] In some embodiments, this disclosure provides a purified capsular polysaccharide comprising one or more units of Formula I, and wherein said purified capsular polysaccharide is conjugated to a carrier protein. In some embodiments, the carrier protein is a fusion protein comprising an antigenic polypeptide (e.g., one described herein, such as SP0785, SP1500, SP0435, Ply, or as described herein, or in WO 05 / 76696, WO 05 / 108580, or WO 10 / 71986, etc.) and / or comprising one or more antigenic polypeptides (e.g., one described herein, including, for example, CP1, SPP2, etc., as described herein). In some embodiments, the carrier protein is a toxoid or a glycoprotein derived from a pathogen. In some embodiments, the carrier protein is or comprises PspA as described in WO 19 / 167008; CRM197 as described in WO 14 / 92378; variants of CRM197 disclosed in WO 22 / 178015, such as eCRM197; diphtheria toxoid; variants of diphtheria toxoid as described in WO 18 / 156465; tetanus toxoid as described in WO 19 / 152921; exoprotein A and protein D as described in US5858677, and combinations thereof.

[0106] In some embodiments, this disclosure provides a purified capsular polysaccharide comprising repeating units of formula I, wherein the purified capsular polysaccharide is conjugated with biotin.

[0107] In some embodiments, activation of the purified capsular polysaccharide of Formula I yields a polysaccharide having one or more monomers of Formula II: II

[0108] In some embodiments, this disclosure provides a polysaccharide having one or more monomers of formula II: II

[0109] In some embodiments, a polysaccharide comprising a monomer of formula II further comprises a monomer of formula I: I Each R is independently selected from H and –C(O)-CH3.

[0110] In some embodiments, this disclosure provides a polysaccharide comprising one or more monomers of formula III: III One of the R a It is CN or -C(NH)NR b , and R among them b It is a protein (e.g., a carrier protein) or biotin, and the rest of the R a Each is H.

[0111] In some embodiments, this disclosure provides a polysaccharide comprising one or more monomers of formula IV-1: IV-1 One of the R a It is -C(NH)NR b , and R b It is biotin or a carrier protein, and the rest of the R a Each is H.

[0112] In some embodiments, this disclosure provides a polysaccharide comprising one or more monomers of formula IV-2: IV-2 One of the R c These are the sites that react with oxidants (e.g., periodate), and the remaining R... c Each of them is -OH.

[0113] In some embodiments, this disclosure provides a polysaccharide comprising one or more monomers of formula IV-3. IV-3 One of the R c These are the sites that react with oxidants (e.g., periodate), and the remaining R... c It is -OH.

[0114] In some embodiments, the weight-average molecular weight of the polysaccharide is about 300 kDa to about 4000 kDa. In some embodiments, the weight-average molecular weight of the polysaccharide is about 600 kDa to about 4000 kDa. In one embodiment, the weight-average molecular weight of the polysaccharide is about 600 kDa. In another embodiment, the weight-average molecular weight of the polysaccharide is about 3,550 kDa.

[0115] In some embodiments, the purified capsular polysaccharide of Formula I is activated by an oxidizing agent. In some embodiments, the oxidizing agent is 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP), periodate (e.g., sodium periodate), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). In some embodiments, the purified capsular polysaccharide of Formula I is activated by reductive ammoniation. In some embodiments, the purified capsular polysaccharide is activated by contacting it with an oxidizing agent. In some embodiments, the oxidizing agent is sodium periodate. In some embodiments, the purified capsular polysaccharide is activated by reductive ammoniation. In some embodiments, the purified capsular polysaccharide is activated by contacting it with a reducing agent. In some embodiments, the reducing agent is sodium cyanoborohydride.

[0116] In some embodiments, activation occurs at the terminal hydroxyl group of the purified capsular polysaccharide.

[0117] In some embodiments, the purified capsular polysaccharide of Formula I is represented by the following formula.

[0118] Other antigenic polysaccharides

[0119] In some embodiments, the immunogenic agents described herein are or contain one or more antigenic polysaccharides (different from those described herein). Streptococcus pneumoniae Purified capsular polysaccharide of serotype 38 (e.g., purified capsular polysaccharide of formula I), or other types besides it.

[0120] In some implementations, the antigenic polysaccharide is or is derived from Streptococcus pneumoniae In some embodiments, one or more antigenic polysaccharides are or are derived from Streptococcus pneumoniae The 1, 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 serotypes or strains (variants). In some embodiments, one or more antigenic polysaccharides are or are derived from more than 25. Streptococcus pneumoniae The serotypes or strains (variants), for example, 26, 27, 28, 29, 20, 35, 40, 45, or 50 serotypes or strains. In some embodiments, one or more antigenic polysaccharides are or are derived from more than 60, 70, 80, 90, or 100. Streptococcus pneumoniae Serotype or strain (variant)

[0121] In some implementations, the antigenic polysaccharide is a branched polysaccharide, or alternatively, a linear polysaccharide.

[0122] In some implementations, the antigenic polysaccharide is or is derived from Streptococcus pneumoniae Polysaccharides. In some embodiments, the antigenic polysaccharides present in the immunogenic agents described herein include a... Streptococcus pneumoniae Polysaccharides. Capsular polysaccharides are used for differentiation. Streptococcus pneumoniae There are at least 100 different serotypes. Streptococcus pneumoniae Polysaccharide serotypes, each with a different chemical structure.

[0123] In some embodiments, the antigenic polysaccharides present in the immunogenic agent include one or more types. pneumococcus bacteria Capsular polysaccharides, derived from or derived from one or more selected from the following Streptococcus pneumoniae Serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B ,11C,11D,11E,11F,12A,12B,12F,13,14,15A,15B,15C,15F,16A,16F,17A,17F,18A,18B,18C,18F,19A,1 9B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

[0124] In some embodiments, the antigenic polysaccharides present in the antigenic agent include one or more types. Streptococcus pneumoniae Capsular polysaccharides, derived from or derived from one or more selected from the following Streptococcus pneumoniae Serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B.

[0125] Antigen polypeptide

[0126] In some embodiments, the immunogenic agents described herein are or contain one or more polypeptide antigens.

[0127] In some embodiments, the polypeptide antigen is or comprises a bacterial polypeptide antigen. In some embodiments, the bacterial polypeptide antigen is a polypeptide antigen of or derived from Gram-negative or Gram-positive bacteria. In some embodiments, the bacterial polypeptide antigen is... Streptococcus pneumoniae The polypeptide antigen or the polypeptide antigen derived from it.

[0128] In some implementations, the polypeptide antigen is Streptococcus pneumoniae The polypeptide antigen or polypeptide antigen derived therefrom. In some embodiments, the immunogenic agent includes one or more of the following: Streptococcus pneumoniae Antigenic polypeptide or a portion thereof.

[0129] Example pneumococcal polypeptide antigen: pneumococcal hemolysin polypeptide

[0130] In some embodiments, the antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or comprises a pneumococcal hemolysin polypeptide antigen. Pneumococcal hemolysin (Ply) is... Streptococcus pneumoniaePly is a cholesterol-dependent toxin belonging to the thiol-activated hemolysin family. In some embodiments, the Ply polypeptide antigen is or comprises a full-length Ply polypeptide. For example, in some embodiments, the full-length Ply polypeptide has 470 amino acids (53 kDa) and is represented by the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the Ply polypeptide antigen comprises a portion of the Ply polypeptide (e.g., a portion of the Ply polypeptide of SEQ ID NO: 4, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids of SEQ ID NO: 4). In some embodiments, a portion of the Ply polypeptide corresponds to a portion of amino acids 2-470 having the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the Ply polypeptide antigen comprises one or more amino acid alterations (e.g., deletions, substitutions, and / or insertions) of a naturally occurring wild-type Ply polypeptide sequence. For example, the Ply polypeptide antigen may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same amino acid sequence as SEQ ID NO: 4, or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids of the sequence shown in SEQ ID NO: 4). Alternatively, the Ply polypeptide antigen may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of a portion of the same sequence as SEQ ID NO: 4 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids). In some embodiments, the nucleotide sequence encoding the Ply polypeptide is provided herein as SEQ ID NO: 14.

[0131] Pneumococcal hemolysin is an exotoxin produced by bacteria that can induce hemolytic activity and complement activation. Despite its high immunogenicity, its use in vaccines may be limited because it causes erythrocyte lysis. Therefore, in some embodiments, the Ply polypeptide antigen is or contains a Ply variant (also referred to herein as mutant Ply or “mPly”) that is substantially non-hemolytic. As used herein, the phrase “substantially non-hemolytic” means a reduction in the ability to lyse erythrocytes of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or up to 100% compared to an equivalent concentration of reference Ply (e.g., wild-type Ply). In some embodiments, the hemolytic activity of the substantially non-hemolytic Ply is at least 5%, at least 10%, at least 15%, at least 20%, at least 20%, at least 30%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% lower than the equivalent concentration of a reference Ply (e.g., wild-type Ply). In some embodiments, the substantially non-hemolytic Ply has no detectable hemolytic activity. The term "wild-type Ply" has the common definition corresponding to the phrase, for example, in some embodiments, it is a naturally occurring Ply (e.g., Ply naturally secreted by a capable bacterial source). In some embodiments, the wild-type Ply protein is represented by the amino acid sequence shown in SEQ ID NO: 4.

[0132] In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) comprises the wild-type Ply amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 4) or an antigenic fragment thereof, having one or more amino acid substitutions. In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) comprises the wild-type Ply amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 4) or an antigenic fragment thereof, having one or more of the following amino acid substitutions: residue D385 is substituted with N; residue C428 is substituted with G; and residue W433 is substituted with F. See, for example, Berry et al., “Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae". Infect Immune 1995 63(5):1969-1974. In some embodiments, the mutant Ply carrying the amino acid substitutions D385N, C428G, and W433F (e.g., non-hemolytic Ply) is referred to as PdT. In some embodiments, PdT is or contains the amino acid sequence as shown in SEQ ID NO: 5.

[0133] In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) comprises the wild-type Ply amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 4) or an antigenic fragment thereof, with G294 substituted with P (see, for example, Oloo et al., “Structure-guided antigen engineering yields pneumolysin mutants suitable for vaccination against pneumococcal disease”. J Biol Chem. 2011 286(14):12133-12140). In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) comprises the wild-type Ply amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 4) or an antigenic fragment thereof, having all of the following amino acid substitutions: residue D285 substituted with N; residue C428 substituted with G; residue W433 substituted with F; and G294 substituted with P. In some embodiments, a mutant Ply carrying the amino acid substitutions G294P, D385N, C428G, and W433F (e.g., non-hemolytic Ply) is referred to as PdT(G294P). In some embodiments, PdT(G294P) is or contains the amino acid sequence as shown in SEQ ID NO: 6.

[0134] In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) is a portion of the PdT(G294P) polypeptide (e.g., a portion of the PdT(G294P) polypeptide of SEQ ID NO: 6, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids of SEQ ID NO: 6). In some embodiments, such a portion of the PdT(G294P) polypeptide comprises the four amino acid substitutions described herein. In some embodiments, a portion of the PdT(G294P) polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) comprises one or more amino acid alterations (e.g., deletions, substitutions, and / or insertions) of the PdT(G294P) polypeptide sequence of SEQ ID NO: 6. For example, a mutant Ply (e.g., non-hemolytic Ply) may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same amino acid sequence as SEQ ID NO: 6, or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids). Alternatively, the mutant Ply (e.g., non-hemolytic Ply) may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same sequence as SEQ ID NO: 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids).In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) may contain no more than 25 amino acid modifications (e.g., deletions, substitutions, and / or insertions) in the sequence or a portion thereof as described herein, SEQ ID NO: 6. In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions.

[0135] Example pneumococcal polypeptide antigen: SP0435 polypeptide

[0136] In some embodiments, the antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or comprises the SP0435 polypeptide. SP0435 is... Streptococcus pneumoniaeProtein. In some embodiments, the SP0435 polypeptide is an elongation factor P. In some embodiments, the SP0435 polypeptide is or comprises the full-length SP0435 polypeptide. For example, in some embodiments, the full-length SP0435 polypeptide has 186 amino acids (20 kDa) and is represented by the amino acid sequence as shown in SEQ ID NO: 8. Not wishing to be bound by any particular theory, amino acids 1-61 of SEQ ID NO: 8 are predicted to be dimerization domains of the SP0435 polypeptide (amino acids 1-61 of the full-length protein). Therefore, in some embodiments, the SP0435 polypeptide may exclude this dimerization domain, for example, in some embodiments, to minimize or avoid the possibility of cross-linking or interfering with the rhizobiamic avidin moiety. In some embodiments, the SP0435 polypeptide comprises a portion of the SP0435 polypeptide (e.g., a portion of the SP0435 polypeptide of SEQ ID NO: 8, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more consecutive amino acids of SEQ ID NO: 8). In some embodiments, a portion of the SP0435 polypeptide corresponds to a protein having amino acids 62-185 of the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the SP0435 polypeptide comprises one or more amino acid alterations (e.g., deletions, substitutions, and / or insertions) of the naturally occurring wild-type SP0435 polypeptide sequence. For example, the SP0435 polypeptide may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same amino acid sequence as SEQ ID NO: 8, or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 70, 75, 80, 85, 90, 95, 100, 150 or more consecutive amino acids of the sequence shown in SEQ ID NO: 8). Alternatively, the SP0435 polypeptide may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of a portion of the same sequence as SEQ ID NO: 8 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more consecutive amino acids).In some embodiments, the nucleotide sequence encoding the SP0435 polypeptide is provided herein as SEQ ID NO: 17. In some embodiments, the SP0434 polypeptide may contain no more than 25 (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid modifications (e.g., deletions, substitutions, and / or insertions) in the sequence or a portion thereof as described herein as in SEQ ID NO: 8. In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions. In some embodiments, the SP0435 polypeptide is one described in WO 2011 / 112906, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0137] Example pneumococcal polypeptide antigen: SP0785 polypeptide

[0138] In some embodiments, the antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or comprises the SP0785 polypeptide. SP0785 is a conservative hypothetical value. pneumococcus bacteria Proteins, for example, in some embodiments described in WO 2014 / 124228, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the SP0785 polypeptide is a protein that... Streptococcus pneumoniaeConserved efflux transport proteins between strains. In some embodiments, the SP0785 polypeptide is or comprises the full-length SP0785 polypeptide. For example, in some embodiments, the full-length SP0785 polypeptide has 299 amino acids (38 kDa) and is represented by the amino acid sequence as shown in SEQ ID NO: 10. Not wishing to be bound by any particular theory, amino acids 1-32 of SEQ ID NO: 10 are predicted to be the signal sequence and transmembrane domain of the SP0785 polypeptide (amino acids 1-32 of the full-length protein). Therefore, in some embodiments, the SP0785 polypeptide may exclude such a signal sequence and transmembrane domain. In some embodiments, the SP0785 polypeptide comprises a portion of the SP0785 polypeptide (e.g., a portion of the SP0785 polypeptide of SEQ ID NO: 10, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or more consecutive amino acids of SEQ ID NO: 10). In some embodiments, a portion of the SP0785 polypeptide corresponds to a protein having amino acids 33-399 of the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the SP0785 polypeptide comprises one or more amino acid alterations (e.g., deletions, substitutions, and / or insertions) of the naturally occurring wild-type SP0785 polypeptide sequence. For example, the SP0785 polypeptide may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same sequence as SEQ ID NO: 10 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or more consecutive amino acids as shown in SEQ ID NO: 10). In some embodiments, the nucleotide sequence encoding the SP0785 polypeptide is provided herein as SEQ ID NO: 18. In some embodiments, the SP0785 peptide may contain no more than 25 (e.g., no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2) amino acid modifications (e.g., deletions, substitutions, and / or insertions) in the sequence of SEQ ID NO: 10 as described herein or a portion thereof.In some embodiments, this amino acid modification may be present in the N-terminal portion and / or the C-terminal portion.

[0139] Example pneumococcal polypeptide antigen: SP1500 polypeptide

[0140] In some embodiments, the antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or comprises an SP1500 polypeptide. SP1500 is a... Streptococcus pneumoniae Proteins, for example, in some embodiments described in WO 2014 / 124228, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the SP1500 polypeptide is an amino acid ABC transporter protein, which is... pneumococcus bacteriaA conserved amino acid-binding polypeptide between strains. In some embodiments, the SP1500 polypeptide is or comprises the full-length SP1500 polypeptide. For example, in some embodiments, the full-length SP1500 polypeptide has 278 amino acids (28 kDa) and is represented by the amino acid sequence shown in SEQ ID NO: 12. Not wishing to be bound by any particular theory, amino acids 1-26 of SEQ ID NO: 12 are predicted to be the signal sequence of the SP1500 polypeptide (amino acids 1-26 of the full-length protein). Therefore, in some embodiments, the SP1500 polypeptide may exclude this signal sequence. In some embodiments, the SP1500 polypeptide comprises a portion of the SP1500 polypeptide (e.g., a portion of the SP1500 polypeptide of SEQ ID NO: 12, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive amino acids of SEQ ID NO: 12). In some embodiments, a portion of the SP1500 polypeptide corresponds to a protein having amino acids 27-278 of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the SP1500 polypeptide comprises one or more amino acid alterations (e.g., deletions, substitutions, and / or insertions) of the naturally occurring wild-type SP1500 polypeptide sequence. For example, the SP1500 polypeptide may contain at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the same sequence as SEQ ID NO: 12 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive amino acids). Alternatively, the SP1500 polypeptide may contain at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) of the same sequence as SEQ ID NO: 12 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive amino acids).In some embodiments, the nucleotide sequence encoding the SP1500 polypeptide is provided herein as SEQ ID NO: 19. In some embodiments, the SP1500 polypeptide may contain no more than 25 (e.g., no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid modifications (e.g., deletions, substitutions, and / or insertions) in the sequence or a portion thereof as described herein as in SEQ ID NO: 12. In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions.

[0141] In some embodiments, the nucleic acid sequences encoding the pneumococcal hemolysin polypeptide (SEQ ID NO: 4), SP0435 polypeptide (SEQ ID NO: 8), SP0785 polypeptide (SEQ ID NO: 10), and SP1500 polypeptide (SEQ ID NO: 12) are provided as SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. Due to the degeneracy of the genetic code, those skilled in the art will understand that other DNA sequences, including codon-optimized sequences, can encode these polypeptides, as well as other polypeptides disclosed herein.

[0142] In some embodiments, this disclosure includes nucleic acid sequences encoding any of the amino acids described herein. Due to the degeneracy of the genetic code, those skilled in the art will understand that other DNA sequences, including codon-optimized sequences, can encode these polypeptides, as well as other polypeptides disclosed herein.

[0143] Fusion proteins containing antigenic peptides

[0144] In some embodiments, the immunogenic agents described herein are or comprise fusion proteins. In some embodiments, the antigenic peptides described herein may be part of a fusion protein. For example, in some embodiments, the immunogenic agents described herein comprise fusion proteins, which are or comprise complementary affinity molecules and one or more antigenic peptides described herein. In some embodiments, the fusion protein comprises two antigenic peptides described herein. In some embodiments, the fusion protein comprises three antigenic peptides described herein. In some embodiments, the fusion protein comprises four antigenic peptides described herein. In some embodiments, the fusion protein comprises 6-10 antigenic peptides described herein. In some embodiments, the fusion protein comprises 11-15 antigenic peptides described herein. In some embodiments, the fusion protein comprises 15-20 antigenic peptides described herein.

[0145] In some embodiments, the fusion protein has carrier properties. In some embodiments, the fusion protein has antigen properties. In some embodiments, the fusion protein has both carrier and antigen properties.

[0146] In some embodiments, the fusion protein comprises an antigenic polypeptide, which is or contains Streptococcus pneumoniae Polypeptide antigens (e.g., in some embodiments, pneumococcal hemolysin polypeptide antigen, SP0435 polypeptide antigen, SP0785 polypeptide antigen, or SP1500 polypeptide antigen).

[0147] In some embodiments, the fusion protein comprises Streptococcus pneumoniae One or more antigenic polypeptides having an amino acid sequence or antigen fragment identical to any one of SEQ ID NO: 4-13 by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the fusion protein comprises... pneumonia chain cocci The fusion protein comprises one or more antigenic polypeptides having an amino acid sequence or antigenic fragment thereof comprising any one of SEQ ID NO: 4-13. In some embodiments, the fusion protein comprises two antigenic polypeptides having an amino acid sequence or antigenic fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 4-13. In some embodiments, the fusion protein comprises two antigenic polypeptides having an amino acid sequence or antigenic fragment thereof comprising any one of SEQ ID NO: 4-13. In some embodiments, the fusion protein described herein further comprises a biotin-binding portion, for example, in some embodiments, the biotin-binding portion is or comprises rhizobialin having an amino acid sequence or biotin-binding fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the fusion protein as described herein further comprises a biotin-binding portion, for example, in some embodiments, the biotin-binding portion is or comprises rhizobiatic avidin having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or its biotin-binding fragment.

[0148] In some embodiments, the fusion protein comprises a pneumococcal hemolysin polypeptide as described herein. For example, in some embodiments, the fusion protein comprises a polypeptide containing the sequence of SEQ ID NO: 7 ( Streptococcus pneumoniae The PdT (G294P) polypeptide (amino acids 2-470) has an amino acid sequence or antigen fragment of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity. In some embodiments, the pneumococcal hemolysin polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein comprises a rhizobiatic peptide, for example, in some embodiments, the rhizobiatic peptide has an amino acid sequence of any one of SEQ ID NO: 1-3 or its biotin-binding fragment.

[0149] In some embodiments, the fusion protein comprises the SP0435 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide containing the sequence of SEQ ID NO: 9 ( Streptococcus pneumoniae The SP0435 polypeptide (amino acids 62-185) has an amino acid sequence or antigen fragment of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity. In some embodiments, the SP0435 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rhizobiamic peptide, for example, in some embodiments, the rhizobiamic peptide has an amino acid sequence of any one of SEQ ID NO: 1-3 or its biotin-binding fragment.

[0150] In some embodiments, the fusion protein comprises the SP0785 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide containing the sequence of SEQ ID NO: 11 ( Streptococcus pneumoniae The SP0785 polypeptide (amino acids 33-399) has an amino acid sequence or antigen fragment thereof with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity. In some embodiments, the SP0785 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rhizobiamic peptide, for example, in some embodiments, the rhizobiamic peptide has an amino acid sequence of any one of SEQ ID NO: 1-3 or its biotin-binding fragment.

[0151] In some embodiments, the fusion protein comprises the SP1500 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide containing the sequence of SEQ ID NO: 13 ( Streptococcus pneumoniae The SP1500 polypeptide (amino acids 27-278) has an amino acid sequence or antigen fragment of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity. In some embodiments, the Sp1500 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rhizobiamic peptide, for example, in some embodiments, the rhizobiamic peptide has an amino acid sequence of any one of SEQ ID NO: 1-3 or its biotin-binding fragment.

[0152] In some embodiments, the fusion protein comprises one or more polypeptides homologous to the polypeptides described herein (e.g., antigenic polypeptides isolated from different serotypes, strains, or species). A single serotype, strain, or species may contain many related mutations, some of which may result in protein sequence differences between different serotypes, strains, or species. In some embodiments, the fusion protein comprises polypeptides homologous to the polypeptides described herein. Streptococcus pneumoniae One or more polypeptides homologous to each other, for example, from different Streptococcus pneumoniae The serotype-isolated Ply polypeptide, SP0435 polypeptide, SP0785 polypeptide, or SP1500 polypeptide. In some embodiments, the antigenic polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with any polypeptide or antigen fragment of any of SEQ ID NO: 4-13. Serotype, strain, or species variations can be used to design such variants of the antigenic polypeptide described herein.

[0153] In some embodiments, the fusion protein described herein comprises one or more fragments of one or more polypeptides described herein, such as a biotin-binding fragment of rhizobiatic avidin or an antigenic fragment of an antigenic polypeptide (e.g., a Ply polypeptide or an antigenic fragment thereof, a Sp0435 polypeptide or an antigenic fragment thereof, a SP0785 polypeptide or an antigenic fragment thereof (e.g., with or without a signal sequence) or a SP1500 polypeptide or an antigenic fragment thereof (e.g., with or without a signal sequence)). In some embodiments, the fusion protein described herein comprises a truncated form of the polypeptide described herein, the truncated form being approximately dimensionally close to a polypeptide having an amino acid sequence of any one of SEQ ID NO: 1-13. For example, in some embodiments, such a truncated polypeptide may be missing up to one, two, three, four, five, ten, or twenty amino acids from one or both ends of its parent polypeptide. In some embodiments, the same number of residues are removed from the N-terminus and C-terminus, while in other embodiments, a different number of residues are removed from the N-terminus compared to the C-terminus. In some embodiments, the truncated polypeptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1-13, but lacks 1-15, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both ends. In some embodiments, the truncated polypeptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1-13, but lacks 1-10 amino acid residues from the N-terminus, C-terminus, or both ends. In some embodiments, the truncated polypeptide has the amino acid sequence of any one of SEQ ID NO: 1-13, lacking 1-10 amino acid residues from the N-terminus, C-terminus, or both. For example, the truncated polypeptide may lack 10 amino acid residues from both the N-terminus and C-terminus of any one of SEQ ID NO: 1-13, resulting in a 20-amino acid residue deficiency in the protein. Internal deletions are also involved, for example, 1-10, 11-20, 21-30, or 31-40 amino acids.

[0154] In some embodiments, the fusion protein comprises an N-terminal polypeptide and a C-terminal polypeptide. In some embodiments, one or both of the N-terminal and C-terminal polypeptides are antigenic polypeptides described herein (e.g., polypeptides having an amino acid sequence comprising one or more of SEQ ID NO: 4-13) or their antigenic fragments or variants. In some embodiments, one or both of the N-terminal and C-terminal polypeptides are biotin-binding moieties, for example, polypeptides having an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or their biotin-binding fragments. In some embodiments, one of the N-terminal or C-terminal polypeptides is a biotin-binding moieties, for example, polypeptides having an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or their biotin-binding fragments, and the other terminal polypeptide is an antigenic polypeptide described herein (e.g., a polypeptide having an amino acid sequence comprising one or more of SEQ ID NO: 4-13) or its antigenic fragments or variants.

[0155] In some embodiments, the N-terminal and C-terminal peptides present in the fusion protein bind directly to each other. In some embodiments, the N-terminal and C-terminal peptides present in the fusion protein are linked by a linker peptide (e.g., the linker disclosed herein). When present, the length and / or amino acids of the linker can be adjusted to obtain a linker with higher flexibility, semi-rigidity, or rigidity. In some embodiments, the linker may be a GS-enriched linker. In some embodiments, the linker may be an A-enriched linker. The length of the linker can typically be 1-40 amino acids, such as 3-10 or 10-30, and particularly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Exemplary flexible peptide linkers are shown in SEQ ID NO: 30-37.

[0156] In some embodiments, the fusion protein comprises one or more linkers described herein. In some embodiments, the fusion protein comprises at least one linker. In some embodiments, the fusion protein comprises at least two linkers. In some embodiments, the linker comprises a polypeptide containing the amino acid sequence GGGGSSS (SEQ ID NO: 30). In some embodiments, the linker comprises a polypeptide containing the amino acid sequence AAA. In some embodiments, the linker comprises a polypeptide containing the amino acid sequence AAAA (SEQ ID NO: 58). In some embodiments, the linker comprises a polypeptide containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 33). In some embodiments, the fusion protein comprises a first linker and a second linker, the first linker having the amino acid sequence GGGGSSS (SEQ ID NO: 30) and the second linker having the amino acid sequence AAA. In some embodiments, the linker sequence may be a residual amino acid sequence from a restriction site. For example, in some embodiments, the fusion protein comprises the amino acid sequence AAA residues from a Not I restriction site. In some embodiments, the fusion protein comprises the linker GGGGSSS (SEQ ID NO: 30) and the amino acid sequence AAA residues from a Not I restriction site. In some embodiments, one or more adapters are selected from GGGGSSS (SEQ ID NO:30) and AAAA (SEQ ID NO:58). In some embodiments, the fusion protein comprises adapters GGGGSSS (SEQ ID NO:30) and AAAA (SEQ ID NO:58).

[0157] In some embodiments, the fusion protein described herein includes a biotin-binding moiety. In some embodiments, the fusion protein includes a biotin-binding moiety and one or more polypeptide antigens. In some embodiments, the fusion protein includes a biotin-binding moiety and two or more polypeptide antigens. In some embodiments, the biotin-binding moiety of the fusion protein includes rhizobiacin or a biotin-binding fragment thereof, which in some embodiments is further described as in WO2012 / 155053, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0158] In some embodiments, the fusion protein described herein includes a biotin-binding portion, which is or comprises a polypeptide or its biotin-binding fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin). In some embodiments, the fusion protein includes a biotin-binding portion, which is or comprises a polypeptide or its biotin-binding fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizobium avidin, referred to as Rhavi) or SEQ ID NO: 3.

[0159] In some embodiments, the fusion egg described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide or a biotin-binding fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin); (ii) a polypeptide comprising a biotin-binding fragment of SEQ ID NO: 7 (rhizobium avidin). Streptococcus pneumoniae The amino acid sequence of the PdT(G294P) polypeptide (amino acid 2-470) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment; and (iii) a polypeptide comprising an amino acid sequence or its antigen fragment that is identical to SEQ ID NO: 9 ( lung Streptococcus inflammatoryae The sequence of amino acids 62-185 of the SP0435 polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment. In some embodiments, the fusion protein further comprises one or more adapters. In some embodiments, such one or more adapters are independently selected from the amino acid sequences of any one of SEQ ID NO: 30-59 and AAA. In some embodiments, such one or more adapters are all GGGGSSS (SEQ ID NO: 30).

[0160] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide or a biotin-binding fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin); (ii) a polypeptide comprising a biotin-binding fragment of SEQ ID NO: 11 (rhizobium avidin). Streptococcus pneumoniae The sequence of the polypeptide SP0785 (amino acids 33-399) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment; and (iii) a polypeptide comprising an amino acid sequence or its antigen fragment that is identical to SEQ ID NO:13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of the SP1500 polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment. In some embodiments, the fusion protein further comprises one or more adapters. In some embodiments, such one or more adapters are independently selected from the amino acid sequences of any one of SEQ ID NO: 30-59 and AAA. In some embodiments, such one or more adapters are selected from GGGGSSS (SEQ ID NO: 30) and AAA. In some embodiments, the fusion protein comprises the amino acid sequence AAA residue from the Not I restriction site. In some embodiments, the fusion protein comprises the adapter GGGGSSS (SEQ ID NO: 30) and the amino acid sequence AAA residue from the Not I restriction site.

[0161] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide or a biotin-binding fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (rhizobium avidin, amino acids 45-179, referred to as Rhavi); (ii) a polypeptide comprising the same sequence as SEQ ID NO: 7 ( Streptococcus pneumoniaeThe amino acid sequence of the PdT(G294P) polypeptide (amino acid 2-470) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment; and (iii) a polypeptide comprising an amino acid sequence or its antigen fragment that is identical to SEQ ID NO: 9 ( Streptococcus pneumoniae The sequence of amino acids 62-185 of the SP0435 polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment. In some embodiments, the fusion protein further comprises one or more adapters. In some embodiments, such one or more adapters are independently selected from the amino acid sequences of any one of SEQ ID NO: 30-59 and AAA. In some embodiments, such one or more adapters are all GGGGSSS (SEQ ID NO: 30). In some embodiments, the fusion protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO: 20 (SPP2) or SEQ ID NO: 21.

[0162] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide or its biotin-binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (rhizobium avidin, amino acids 45-179, referred to as Rhavi); (ii) a polypeptide comprising the same sequence as SEQ ID NO: 11 ( Streptococcus pneumoniae The sequence of the polypeptide SP0785 (amino acids 33-399) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment; and (iii) a polypeptide comprising an amino acid sequence or its antigen fragment identical to SEQ ID NO: 13 ( Streptococcus pneumoniaeThe sequence of amino acids 27-278 of the SP1500 polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment. (iii) A polypeptide comprising an amino acid sequence or its antigen fragment identical to SEQ ID NO: 13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of the SP1500 polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigen fragment. In some embodiments, such one or more adapters are independently selected from the amino acid sequences of any one of SEQ ID NO: 30-59 and AAA. In some embodiments, such one or more adapters are selected from GGGGSSS (SEQ ID NO: 30) and AAA. In some embodiments, the fusion protein comprises the amino acid sequence AAA residue from the Not I restriction site. In some embodiments, the fusion protein comprises the adapter GGGGSSS (SEQ ID NO: 30) and the amino acid sequence AAA residue from the Not I restriction site. In some embodiments, the fusion protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the fusion protein is composed of the amino acid sequence SEQ ID NO: 24 (CP1) or SEQ ID NO: 25.

[0163] In some embodiments, this disclosure provides a fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with a fusion protein having the amino acid sequence of any one of SEQ ID NO: 20-25. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SPP2. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with CP1.

[0164] In some embodiments, the fusion protein described herein comprises an antigenic fragment of a fusion protein having the amino acid sequence of any one of SEQ ID NO: 20-25. For example, the fusion protein may be missing at least one, two, three, four, five, ten, or twenty amino acids from the N-terminus, C-terminus, or both of SEQ ID NO: 20-25. In some embodiments, the same number of residues are removed from the N-terminus and C-terminus, while in other embodiments, a different number of residues are removed from the N-terminus compared to the C-terminus. In some embodiments, the fusion protein is or comprises the antigenic fragment of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein is or comprises the antigenic fragment of SPP2. In some embodiments, the fusion protein is or comprises the antigenic fragment of SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the fusion protein is or comprises the antigenic fragment of CP1.

[0165] In some embodiments, the fusion protein described herein comprises a variant or fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 1-13. In some embodiments, the fusion protein described herein comprises a polypeptide encoded by a variant or fragment of a gene having the nucleic acid sequence of SEQ ID NO: 1-13. In some embodiments, the fragment included in the fusion protein described herein is close in size to the full-length polypeptide or a polypeptide having the amino acid sequence of SEQ ID NO: 1-13. For example, it may be missing up to one, two, three, four, five, ten, twenty, or thirty amino acids at one or both ends. In some embodiments, the fragment is 25-50 amino acids long, or 50-100, 100-150, 150-200, 200-250, 250-300, or 300-350 amino acids long. In some embodiments, the fragment is obtained by expressing a host-treated or partially treated signal sequence, for example, E. coli Insect cell lines (e.g., baculovirus expression systems), yeast (e.g., S. cerevisiae or S. pombe Cell lines or mammalian (e.g., human or Chinese hamster ovary) cell lines. The fragments described above or their sub-fragments (e.g., fragments of 8-50, 8-30 or 8-20 amino acid residues) preferably have one of the biological activities described below, such as increasing the amount of released IL-17 by at least 1.5-fold or 2-fold or more (e.g., whether as an absolute indicator or relative to a control protein).

[0166] In some embodiments, the fusion protein described herein can be used in the immunogenic agents described herein.

[0167] Connector or spacer

[0168] In some embodiments, one or more adapters are used to couple the polypeptides described herein to one or more entities of interest (e.g., small molecules, polypeptides, polynucleotides, lipids, polysaccharides, etc.). In some embodiments, the fusion protein described herein includes one or more adapters. In some embodiments, the adapter is or contains one or more amino acids. In some embodiments, the fusion protein includes an antigenic polypeptide linked to a biotin-binding moiety via an adapter. In some embodiments, the fusion protein includes a first antigenic polypeptide, a second antigenic polypeptide, a biotin-binding moiety, and at least one adapter. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are linked via adapters. In some embodiments, the first antigenic polypeptide or the second antigenic polypeptide is linked to the biotin-binding moiety via an adapter. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are linked via a first adapter; the first antigenic polypeptide or the second antigenic polypeptide is linked to the biotin-binding moiety via a second adapter.

[0169] In some embodiments, the fusion protein described herein comprises a first adapter comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30) and a second adapter comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30). In some embodiments, the fusion protein described herein comprises a first adapter comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30) and a second adapter comprising the amino acid sequence AAA.

[0170] In some embodiments, the linker inserts a structure between two protein moieties. In some embodiments, the structure is or comprises an α-helix. In some embodiments, the structure is or comprises a β-chain. In some embodiments, the structure is or comprises a helix / bend. In some embodiments, the structure is or comprises a turn. In some embodiments, the linker reduces steric hindrance between the two protein moieties connected by the linker. In some embodiments, the linker reduces adverse interactions between the two protein moieties connected by the linker. In some embodiments, the linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may also comprise threonine, proline, and / or alanine residues. In some embodiments, the linker is hydrophilic. In some embodiments, the linker is hydrophobic. In some embodiments, the linker increases the stability of the fusion protein containing the linker.

[0171] In some embodiments, the linker does not interfere with the folding or activity of the peptide of interest (e.g., an antigenic peptide in some embodiments) to which it is attached. In some embodiments, the linker does not interfere with the antigenicity of the antigenic peptide to which it is attached. In some embodiments, the linker does not reduce the antigenicity of the antigenic peptide to which it is attached. In some embodiments, the linker does not eliminate the antigenicity of the antigenic peptide to which it is attached. In some embodiments, the role of the linker is determined by comparing the peptide to the peptide attached to the linker.

[0172] In some embodiments, the connector does not interfere with the folding of the biotin-binding portion to which it is connected. In some embodiments, the connector does not interfere with the biotin-binding capacity of the biotin-binding portion to which it is connected. In some embodiments, the connector does not reduce the biotin-binding capacity of the biotin-binding portion to which it is connected. In some embodiments, the connector's function is determined by comparing the biotin-binding portion to a biotin-binding portion connected to the connector.

[0173] In some embodiments, the adapter is not antigenic. In some embodiments, the adapter does not elicit a T-cell response. In some embodiments, the adapter does not elicit a B-cell response. In some embodiments, the adapter does not induce a T-cell or B-cell response.

[0174] In some embodiments, the linker may contain two or more amino acids. In some embodiments, the length of the linker may be 3-100, 5-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, or 2-3 amino acids. In some embodiments, the linker comprises 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, or 10-15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, the linker is or comprises a peptide linker comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0175] In some embodiments, the linker is a flexible linker. Flexible linkers can be used to connect domains that require a certain degree of motion or interaction and can include small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and the protein moiety. In some embodiments, the linker comprises small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. In some embodiments, the linker is a Gly-Ser linker.

[0176] In some embodiments, the linker is or contains the amino acid sequence GGGGSSS (SEQ ID NO: 30). In some embodiments, the linker is or contains the sequence (GGGGS). n (SEQ ID NO: 31), where n represents the number of repeating GGGGS (SEQ ID NO: 32) units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the peptide linker may have an amino acid sequence that is or contains GGGGSGGGGSGGGGS (SEQ ID NO: 33) (i.e., (GGGGS)3) or GGGGSGGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 34) ((GGGGS)6). In some embodiments, the linker is or contains (Gly). n(SEQ ID NO: 35), where n represents the number of repeating Gly residues, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or contains GGG (i.e., (Gly)3). In some embodiments, the linker is or contains (Gly)6 (SEQ ID NO: 36). In some embodiments, the linker is or contains (Gly)8 (SEQ ID NO: 37). In some embodiments, the linker contains one or more of Gly, Ser, Thr, Ala, Lys and Glu. In some embodiments, the linker is or contains KESGSVSSEQLAQFRSLD (SEQ ID NO: 38). In some embodiments, the linker is or contains EGKSSGSGSESKST (SEQ ID NO: 39). In some embodiments, the linker is or contains GSAGSAAGSGEF (SEQ ID NO: 40). In some embodiments, the linker is or contains the amino acid sequence AAA. In some embodiments, such a linker may be synthetic or derived from amino acid residues from a restriction site (e.g., the Not I restriction site).

[0177] In some embodiments, the joint is a rigid joint. Rigid joints are used to maintain a fixed distance between structural domains and preserve their independent function. Rigid joints may also be useful when spatial separation of structural domains is critical to maintaining the stability or bioactivity of one or more components in a fusion. In some embodiments, the joint is or comprises (EAAAK). n (SEQ ID NO: 41), where n represents the number of repeating EAAAK (SEQ ID NO: 42) units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the connector is or includes A(EAAAK). n A (SEQ ID NO: 43), where n represents the number of repeating EAAAK (SEQ ID NO: 42) units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the connector is or contains A(EAAAK). nA (SEQ ID NO: 44), where n represents the number of repeating EAAAK (SEQ ID NO: 42) units, and is 2, 3, 4, or 5. In some embodiments, the connector is or contains A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 45). In some embodiments, the connector is or contains [A(EAAAK)... n A] m (SEQ ID NO: 46), where n is 2, 3, or 4 and m is 1 or 2. In some embodiments, the connector is or contains AEAAAAKEAAAKA (SEQ ID NO: 47).

[0178] In some implementations, the connector is or includes (X-Pro). n (SEQ ID NO: 48), where X represents any amino acid, and n represents the number of repeating X-Pro units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or contains (Ala-Pro). n (SEQ ID NO:49), where n represents the number of repeating Ala-Pro units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the connector is or includes (Ala-Pro). n (SEQ IDNO: 50), where n represents the number of repeating Ala-Pro units, and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0179] In some implementations, the connector is or includes (Lys-Pro). n (SEQ ID NO: 51), where n represents the number of repeating Lys-Pro units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the connector is or includes (Glu-Pro)n (SEQ ID NO: 52), where n represents the number of repeating Glu-Pro units, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the connector is or includes (Ala-Pro)7 (SEQ ID NO: 53).

[0180] In some embodiments, the connector is or contains GAGGGGGAAAAAGGGGGGAP (GAG connector, SEQ ID NO: 54). In some embodiments, the connector is or contains GAGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 connector, SEQ ID NO: 55). In some embodiments, the connector is or contains GAGGGGGAAAAAGGGGGGAPGGGGAAAAAGGGGGGAPGGGGAAAAAGGGGGGAP (GAG3 connector, SEQ ID NO: 56).

[0181] In some implementations, the connector is or includes a VSDP (SEQ ID NO: 57).

[0182] In some implementations, the connector is or contains AAAA (SEQ ID NO: 58).

[0183] In some implementations, the connector is or contains GGGG (SEQ ID NO: 59).

[0184] Suitable linkers or spacers also include those having an amino acid sequence that is homologous to or identical to the exemplary linkers described above by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more. In some embodiments, the linker comprises a polypeptide containing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of GGGGSSS (SEQ ID NO: 30).

[0185] Other connectors applicable to some implementations can be found in U.S. Patent Application Publication No. 2012 / 0232021, filed March 2, 2012, and [Chen, 2013], the entire contents of which are incorporated herein by reference.

[0186] Label

[0187] In some embodiments, the polypeptides described herein (e.g., fusion proteins) may include one or more tags. In some embodiments, the fusion proteins described herein include one or more tags. Tags may be present at the N-terminus or C-terminus of the polypeptide. For example, tags may be added to the polypeptide (e.g., by adding to or modifying the coding DNA sequence) to facilitate purification, detection, dissolution, or to impart other desired characteristics to the polypeptide. In some embodiments, the polypeptide may be a peptide, oligopeptide, or polypeptide for affinity purification. In some embodiments, the tag is, contains, or is derived from one or more of the following: polyhistidine (His), glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose-binding protein (MBP), vesicular stomatitis virus glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotinylate carboxyl carrier protein (BCCP), calmodulin, Nus, S-tag, lipoprotein D, and galactosidase. In some embodiments, the His tag is or contains an amino acid sequence of Hn, where n is an integer between 2 and 10 (SEQ ID NO: 60). Exemplary His tags include HHHHHH (SEQ ID NO: 61) and MSYYHHHHHH (SEQ ID NO: 62). In other embodiments, the polypeptides described herein are tagless, such as protein purification tags, and are purified by a method independent of purification tag affinity. In some embodiments, the fusion proteins described herein comprise (i) a polypeptide of any one of the amino acid sequences of SEQ ID NO: 4-13 or a fusion protein of any one of the amino acid sequences of SEQ ID NO: 20-25, and (ii) a tag having no more than 1, 2, 3, 4, 5, 10, or 20 amino acids at one or both ends of the polypeptide or fusion protein of (i).

[0188] In some embodiments, the polypeptides described herein may contain membrane translocation sequences (MTS), for example, in some embodiments, to facilitate the introduction of the fusion protein into mammalian cells and subsequently stimulate a cell-mediated immune response. Exemplary membrane translocation sequences include, but are not limited to, the hydrophobic region of the Kaposi's fibroblast growth factor signaling sequence, the MTS of synuclein, the third helix of the antennal foot homology domain, SN50, the integrin 3h region, HIV Tat, pAntp, PR-39, abaecin (a bee antimicrobial peptide), apidaecin (a bee defense peptide), Bac5, and Bac7. P. berghei CS proteins and those MTSes described in U.S. Patent Nos. 6,248,558; 6,432,680; and 6,248,558.

[0189] In some embodiments, the polypeptides described herein (e.g., fusion proteins) may include one or more tags. In some embodiments, the fusion protein includes one or more tags. The tag may be N-terminal or C-terminal. For example, the tag may be added to the protein (by adding to or modifying the coding DNA sequence) to facilitate purification, detection, dissolution, or to impart other desired characteristics to the protein. In some embodiments, the tag may be a peptide, oligopeptide, or polypeptide that can be used for affinity purification. In some embodiments, the tag is, contains, or is derived from one or more of polyhistidine (His), glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose-binding protein (MBP), vesicular stomatitis virus glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotinylate carboxyl carrier protein (BCCP), calmodulin, Nus, S-tag, lipoprotein D, and galactosidase. In some embodiments, the His tag is or contains the amino acid sequence Hn, where n is an integer between 2 and 10 (SEQ ID NO: 60). Exemplary His tags include HHHHHH (SEQ ID NO: 61) and MSYYHHHHHH (SEQ ID NO: 62). In other embodiments, the fusion proteins described herein are tagless, such as protein purification tags, and are purified by methods independent of purification tag affinity. In some embodiments, the fusion proteins described herein comprise (i) a polypeptide of any one of the amino acid sequences of SEQ ID NO: 1-13 or a fusion protein of any one of the amino acid sequences of SEQ ID NO: 20-25, and (ii) a tag having no more than 1, 2, 3, 4, 5, 10, or 20 amino acids at one or both ends of the polypeptide or fusion protein of (i).

[0190] In some embodiments, the polypeptides described herein may contain membrane translocation sequences (MTS), for example, in some embodiments, to facilitate the introduction of fusion proteins into mammalian cells and subsequently stimulate cell-mediated immune responses. Exemplary membrane translocation sequences include, but are not limited to, the hydrophobic region of the Kaposi's fibroblast growth factor signaling sequence, the MTS of synuclein, the third helix of the antennal foot homology domain, SN50, the integrin 3h region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, the Plasmodium berghei CS protein, and those MTS described in U.S. Patent Nos. 6,248,558; 6,432,680; and 6,248,558.

[0191] signal peptide

[0192] In some embodiments, the polypeptide described herein contains a signal sequence. In some embodiments, the fusion protein contains a signal peptide. The signal peptide, also known as a leader peptide, functions in the translocation of the polypeptide across the cell membrane (e.g., plasma membrane, endoplasmic reticulum, etc.). In some embodiments, the polypeptide contains a signal at or near its N-terminus. In some embodiments, the signal sequence may or may not be cleaved after translocation across the membrane (e.g., by a signal peptidase).

[0193] The signal sequence can be directly linked (e.g., via peptide bonds) or indirectly linked (e.g., via a linker) to the polypeptide. In some embodiments, the signal sequence can be linked to the polypeptide via the peptide linkers disclosed herein.

[0194] In some embodiments, the signal peptide may be a bacterial signal peptide, a viral signal peptide, or a eukaryotic signal peptide. In some embodiments, the eukaryotic signal peptide is a mammalian signal peptide (e.g., a signal peptide derived from or derived from human proteins).

[0195] In some embodiments, the signal peptide may be a bacterial signal peptide. Bacterial signal peptides can be used to promote the translocation of the polypeptide into the bacterial periplasmic space. In some embodiments, the signal peptide translocates into the bacteria (e.g., E. coli The signal peptide is cleaved from the polypeptide after passing through the periplasmic space. Signal peptides secreted directly from proteins in bacterial cells are well known in the art, for example, as disclosed in PCT patent application publication number WO 2005 / 071088.

[0196] Any signal peptide known to those skilled in the art can be used. In some embodiments, the signal peptide is or comprises the following amino acid sequences: MKKIWLALAGLVLAFSASA (SEQ ID NO: 63), MAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 64), MKKVAAFVALSLLMAGC (SEQ ID NO: 65), MKKIMLVITLILVSPIAQQTEAKD (SEQ ID NO: 66), MKKKIISAILMSTVILSAAAPLSGVYADT (SEQ ID NO: 67), MKKRKVLIPLMALSTILVSSTGNLEVIQAEV (SEQ ID NO: 68), MNMKKATIAATAGIAVTAFAAPTIASAST (SEQ ID NO: 69), MQKTRKERILEALQEEKKNKKSKKFKTGATIAGVTAIATSITVPGIEVIVSADE (SEQ ID NO: 70), MKKLKMASCALVAGLMFSGLTPNAFAED (SEQ ID NO: 70). 71), MAKKFNYKLPSMVALTLVGSAVTAHQVQAAE (SEQ ID NO: 72), MTDKKSENQTEKTETKENKGMTRREMLKLSAVAGTGIAVGATGLGTILNVVDQVDKALT (SEQ ID NO: 73), MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLGLGLTIAQSVGAFG (SEQ ID NO: 74), or their derivatives or functional portions. In some embodiments, the signal peptide is or comprises the amino acid sequence of the human Ig heavy chain V-III region VH26 signal peptide or its derivatives or functional portions. In some embodiments, the signal peptide is or comprises the amino acid sequence of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 75), or its derivatives or functional portions. In some embodiments, the signal peptide is or comprises the human IgG2 heavy chain signal peptide or its derivatives or functional portions. In some embodiments, the signal peptide is or contains an amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 76) or a derivative or functional part thereof.

[0197] Other examples of signal peptides can be found on the Signal Peptide website, a database of signal peptides, which can be found at www.signalpeptide.de.

[0198] In some embodiments, the polypeptides described herein lack a signal peptide.

[0199] Affinity molecules

[0200] As described herein, in some embodiments, the immunogenic agents of this disclosure comprise complementary affinity molecule pairs, comprising an affinity molecule (i.e., a first affinity molecule) and a complementary affinity molecule (i.e., a second affinity molecule complementary to the first affinity molecule). In some embodiments, the immunogenic agent comprises a first affinity molecule. In some embodiments, the immunogenic agent comprises a second affinity molecule. In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of the following: biotin / biotin-binding moieties, antibodies / antigens, enzymes / substrates, receptors / ligands, metal / metal-binding proteins, carbohydrate / carbohydrate-binding proteins, lipid / lipid-binding proteins, and His-tag / His-tag-binding molecules. In some embodiments, the first affinity molecule associates with a polymer (e.g., but not limited to, polysaccharides). In some embodiments, the second affinity molecule associates with a peptide of interest (e.g., but not limited to, peptide antigens).

[0201] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is a biotin-binding moiety, such as a biotin-binding protein or polypeptide, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the fusion protein of the immunogenic agent comprises a biotin-binding moiety and one or more polypeptide antigens. In some embodiments, the fusion protein comprises a biotin-binding moiety and two or more antigens. In some embodiments, the biotin-binding moiety is a biotin-binding moiety capable of forming a dimer. In some embodiments, the biotin-binding moiety is a biotin-binding moiety capable of forming a tetramer. In some embodiments, the biotin-binding moiety is or comprises rhizobium avidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrAvidin, or CaptAvidin. TM Or its biotin-binding domain or biotin-binding fragment, or a combination thereof. In some embodiments, the biotin-binding portion is or contains rhizobiacin or its biotin-binding domain or biotin-binding fragment.

[0202] In some embodiments, the immunogenic agents described herein utilize the high affinity between biotin and rhizobium avidin (dissociation constant [KD] ≈ 10). -15M) Non-covalently bound, the rhizobium avidin is a biotin-binding protein predicted to have no significant homology with human proteins. Rhizobium avidin is a naturally occurring dimeric protein in the avidin protein family, originally found in the symbiotic bacterium *Rhizobium ateri* (*Rhizobium ethigrin*), a common legume. Rhizobium etli It was found in [the study]. Rhizobiacin shares only 22% amino acid identity with chicken avidin (a protein commonly found in eggs), but the amino acid residues involved in biotin binding are highly conserved. No cross-reactivity with rhizobiacin was observed in human serum samples obtained from subjects exposed to avidin [Helppolainen et al., 2007], suggesting that rhizobiacin antibodies may not cross-react with chicken avidin. Biotin conjugates have been used in various clinical applications without any reported adverse events [Buller et al., 2014; Paty et al., 2010; Lazzeri et al., 2004].

[0203] In some embodiments, the biotin-binding portion useful according to this disclosure includes rhizobiacin or its biotin-binding domain or biotin-binding fragment, for example, as further described in some embodiments of WO 2012 / 155053, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the biotin-binding portion is or comprises a polypeptide or its biotin-binding domain or biotin-binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with rhizobiacin. In some embodiments, the biotin-binding portion useful according to this disclosure comprises the polypeptide of SEQ ID NO: 1 or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding portion is or comprises a polypeptide or its biotin-binding domain or biotin-binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the biotin-binding portion useful according to this disclosure comprises a polypeptide or its biotin-binding domain or biotin-binding fragment of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the biotin-binding portion is or comprises a polypeptide or its biotin-binding domain or biotin-binding fragment having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 3.

[0204] Fusion protein SPP2

[0205] In some embodiments, the fusion protein described herein is or comprises the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein), and Streptococcus pneumoniae One or more polypeptides or derived from Streptococcus pneumoniae One or more polypeptides. In some embodiments, the provided fusion protein is SPP2, which is further described in International Application No. PCT / US2022 / 043156, filed September 9, 2022, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the fusion protein comprises (i) a biotin-binding moiety (e.g., biotin-binding protein), and (ii) a non-hemolytic pneumococcal hemolysin polypeptide, which is present in wild-type Streptococcus pneumoniae (… Streptococcus pneumoniae (iii) Mutations are found at amino acid residues 294, 385, 428, and 433 of pneumococcal hemolysin or its antigenic fragments; Streptococcus pneumoniae Elongation factor P (SP0435) polypeptide or an antigenic fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a rhizobium avidin polypeptide or its avidin-binding moiety. In some embodiments, the fusion protein comprises (i) the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein), and (ii) a polypeptide comprising the same as SEQ ID NO: 7 ( Streptococcus pneumoniae The amino acid sequence of PdT (G294P) having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence 2-470, or its antigenic variant or fragment, and (iii) a polypeptide containing the amino acid sequence of SEQ ID NO: 9 ( Streptococcus pneumoniaeThe sequence of amino acids 62-185 of SP0435 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or an antigenic variant or fragment thereof. In some embodiments, the fusion protein comprises a polypeptide containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein is or comprises (i) a complementary affinity molecule as described herein (e.g., a biotin-binding moiety as described herein), (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with pneumococcal hemolysin, or an antigenic variant or fragment thereof, and (iii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SP0435, or an antigenic variant or fragment thereof. In some embodiments, the fusion protein is or comprises (i) a complementary affinity molecule as described herein (e.g., a biotin-binding moiety as described herein), (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with, or an antigenic variant or fragment thereof, of a non-hemolytic pneu ...

[0206] In some embodiments, the fusion protein described herein comprises (i) the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein) and (ii) a polypeptide comprising the same as SEQ ID NO: 7 ( Streptococcus pneumoniaeThe amino acid sequence of PdT (G294P) (amino acid 2-470) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence or its antigenic variant or fragment. In some embodiments, the fusion protein comprises (i) the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein) and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 ( Streptococcus pneumoniae The amino acid sequence of SP0435 (amino acids 62-185) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity with an amino acid sequence or its antigenic variant or fragment.

[0207] In some embodiments, the fusion protein described herein comprises a biotin-binding moiety, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin), or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a biotin-binding moiety, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizobium avidin) or SEQ ID NO: 3, or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 7 (rhizobium avidin). Streptococcus pneumoniae The amino acid sequence of PdT (G294P) (amino acid 2-470) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, or its antigenic variant or fragment. In some embodiments, the fusion protein comprises a polypeptide containing amino acids identical to those in SEQ ID NO: 9 ( Streptococcus pneumoniae The amino acid sequence of SP0435 (amino acids 62-185) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, or its antigenic variants or fragments.

[0208] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin), or its biotin-binding fragment; (ii) a polypeptide comprising the same sequence as SEQ ID NO: 7 ( Streptococcus pneumoniae The amino acid sequence of PdT (G294P) having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, or its antigenic variants or fragments; and (iii) a polypeptide comprising an amino acid sequence having the same identity as SEQ ID NO: 9 ( Streptococcus pneumoniae The sequence of amino acids 62-185 of SP0435 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity, or its antigenic variants or fragments. In some embodiments, this fusion protein also includes one or more adapters.

[0209] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding moiety, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizobiatic avidin) or SEQ ID NO: 3, or its biotin-binding fragment; (ii) a polypeptide comprising the same sequence as SEQ ID NO: 7 ( Streptococcus pneumoniae The amino acid sequence of PdT (G294P) having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, or its antigenic variants or fragments; and (iii) a polypeptide comprising an amino acid sequence having the same identity as SEQ ID NO: 9 ( Streptococcus pneumoniae The sequence of amino acids 62-185 of SP0435 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity, or its antigenic variants or fragments. In some embodiments, this fusion protein also includes one or more adapters.

[0210] In some embodiments, the fusion protein described herein is or comprises the SPP2 fusion protein. In some embodiments, the SPP2 fusion protein comprises, from its N-terminus to its C-terminus, (i) a biotin-binding moiety (e.g., a biotin-binding protein), (ii) a first peptide linker, (iii) the pneumococcal hemolysin (Ply) polypeptide described herein or an antigenic variant or fragment thereof; (iv) a second peptide linker, and (v) the SP0435 polypeptide described herein or an antigenic variant or fragment thereof. In some such embodiments, the biotin-binding moiety is or comprises rhizobiastin or its biotin-binding moiety. In some embodiments, the SPP2 fusion protein comprises (i) a biotin-binding portion, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobacterial avidin) or SEQ ID NO: 2 (amino acids 45-179 of rhizobacterial avidin) or SEQ ID NO: 3, or its biotin-binding fragment; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 30); (iii) the pneumococcal hemolysin (Ply) polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 30); and (v) the SP0435 polypeptide described herein. In some embodiments, the SPP2 fusion protein comprises, from the N-terminus to the C-terminus, (i) a biotin-binding portion, which is or comprises a polypeptide of SEQ ID NO:1 (rhizobacterial avidin), SEQ ID NO:2 (amino acids 45-179 of rhizobacterial avidin), or SEQ ID NO:3, or a biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30); (iii) the pneumococcal hemolysin (Ply) polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30); and (v) the SP0435 polypeptide described herein. In some embodiments, such SPP2 fusion proteins may also comprise a detection or purification tag, such as a His tag. In some embodiments, an SPP2 fusion protein comprising a His tag is referred to as SPP2-H. In some such embodiments, the SPP2 fusion protein includes an SP0435 polypeptide (e.g., one described herein) between the biotin-binding moiety and the Ply polypeptide (e.g., one described herein). In some embodiments, the SPP2 fusion protein may include a Ply polypeptide between the biotin-binding moiety and the SP0435 polypeptide.In some embodiments, the Ply polypeptide contained in the fusion protein described herein is or contains the same as SEQ ID NO: 7. pneumonia chain cocci The amino acid sequence of PdT (G294P) (amino acid 2-470) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity, or its antigenic variants or fragments. In some embodiments, the SP0435 polypeptide included in the fusion protein described herein is or contains amino acid sequences identical to those in SEQ ID NO: 9 ( lung Streptococcus inflammatoryae The sequence of amino acids 62-185 of SP0435 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or its antigenic variants or fragments. In some embodiments, the fusion protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein described herein consists of the amino acid sequence SEQ ID NO: 20 or SEQ ID NO: 21.

[0211] fusion protein CP1

[0212] In some embodiments, the fusion protein described herein is or comprises the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein), and Streptococcus pneumoniae One or more polypeptides or derived from Streptococcus pneumoniae One or more polypeptides. In some embodiments, the fusion protein described herein is CP1, which is further described in International Patent Publication No. WO 2020 / 056127, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the fusion protein comprises (i) a biotin-binding moiety (e.g., biotin-binding protein), and (ii) a polypeptide comprising the same as SEQ ID NO: 11 ( Streptococcus pneumoniaeThe amino acid sequence of SP0785 (amino acids 33-399) having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, or its antigenic variant or fragment, and (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 ( Streptococcus pneumoniae The amino acid sequence of SP1500 (amino acids 27-278) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or an antigenic variant or fragment thereof. In some embodiments, the fusion protein comprises a polypeptide containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 25. In some embodiments, the fusion protein is or comprises: (i) a complementary affinity molecule as described herein (e.g., a biotin-binding moiety as described herein), (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SP0785, or an antigenic variant or fragment thereof, and (iii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SP1500, or an antigenic variant or fragment thereof.

[0213] In some embodiments, the fusion protein described herein comprises (i) the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein) and (ii) a polypeptide comprising the same as SEQ ID NO: 11 ( Streptococcus pneumoniae The sequence of amino acids 33-399 of SP0785 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or its antigenic variant or fragment. In some embodiments, the fusion protein comprises (i) the complementary affinity molecule described herein (e.g., the biotin-binding moiety described herein) and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 ( Streptococcus pneumoniaeThe sequence of amino acids 27-278 of SP1500 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity, or its antigenic variant or fragment.

[0214] In some embodiments, the fusion protein described herein comprises a biotin-binding moiety, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin), or its biotin-binding fragment. In some embodiments, the fusion protein comprises a biotin-binding moiety, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizobium avidin) or SEQ ID NO: 3, or its biotin-binding fragment. In some embodiments, the fusion protein comprises a polypeptide containing the same sequence as SEQ ID NO: 11 (rhizobium avidin). pneumonia chain cocci The sequence of amino acids 33-399 of SP0785 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or its antigenic variant or fragment. In some embodiments, the fusion protein comprises a polypeptide containing amino acids identical to those in SEQ ID NO: 13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of SP1500 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity, or its antigenic variant or fragment.

[0215] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin), or its biotin-binding fragment; (ii) a polypeptide comprising the same sequence as SEQ ID NO: 11 (rhizobium avidin). Streptococcus pneumoniaeThe sequence of amino acids 33-399 of SP0785 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or its antigenic variant or fragment; and (iii) a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of SP1500 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity, or its antigenic variants or fragments. In some embodiments, this fusion protein also includes one or more adapters.

[0216] In some embodiments, the fusion protein described herein comprises the following: (i) a biotin-binding portion, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizobiatic avidin) or SEQ ID NO: 3, or its biotin-binding fragment; (ii) a polypeptide comprising the same sequence as SEQ ID NO: 11 ( pneumonia Streptococcus The sequence of amino acids 33-399 of SP0785 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or its antigenic variant or fragment; and (iii) a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of SP1500 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity, or its antigenic variants or fragments. In some embodiments, this fusion protein also includes one or more adapters.

[0217] In some embodiments, the fusion protein described herein is or comprises the CP1 fusion protein. In some embodiments, the CP1 fusion protein comprises, from its N-terminus to its C-terminus, (i) a biotin-binding moiety (e.g., a biotin-binding protein), (ii) a first peptide linker, (iii) the SP1500 polypeptide described herein or an antigenic variant or fragment thereof, (iv) a second peptide linker, and (v) the SP0785 polypeptide described herein or an antigenic variant or fragment thereof. In some such embodiments, the biotin-binding moiety is or comprises rhizobiacin or its biotin-binding moiety. In some embodiments, the CP1 fusion protein comprises (i) a biotin-binding portion, which is or comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (rhizobium avidin) or SEQ ID NO: 2 (amino acids 45-179 of rhizobium avidin) or SEQ ID NO: 3, or a biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30); (iii) the SP0785 polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence AAAaa; and (v) the SP1500 polypeptide described herein. In some embodiments, the CP1 fusion protein comprises, from its N-terminus to its C-terminus, (i) a biotin-binding moiety, which is or comprises a polypeptide of SEQ ID NO: 1 (rhizobacterial avidin), SEQ ID NO: 2 (amino acids 45-179 of rhizobacterial avidin), or SEQ ID NO: 3, or a biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence GGGGSSS (SEQ ID NO: 30); (iii) the SP1500 polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence AAA; and (v) the SP0785 polypeptide described herein. In some embodiments, such CP1 fusion proteins may also comprise a detection or purification tag, such as a His tag. In some such embodiments, the CP1 fusion protein comprises the SP1500 polypeptide between the biotin-binding moiety and the SP0785 polypeptide. In some embodiments, the CP1 fusion protein may comprise the SP0785 polypeptide between the biotin-binding moiety and the SP1500 polypeptide. In some embodiments, the SP0785 peptide included in the fusion protein described herein is or includes a peptide similar to SEQ ID NO: 11 ( Streptococcus pneumoniaeThe sequence of amino acids 33-399 of SP0785 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence, or its antigenic variant or fragment. In some embodiments, the SP1500 polypeptide included in the fusion protein described herein is or contains amino acids identical to those in SEQ ID NO: 13 ( Streptococcus pneumoniae The sequence of amino acids 27-278 of SP1500 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or its antigenic variants or fragments. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

[0218] carrier proteins

[0219] In some embodiments, the immunogenic agents described herein are or comprise one or more carrier proteins. In some embodiments, the carrier protein is or comprises an antigenic polypeptide (e.g., antigenic polypeptides described herein include, for example, SP0785, SP1500, SP0435, Ply as described herein or in WO 05 / 76696, WO 05 / 108580 or WO 10 / 71986) and / or a fusion protein comprising one or more antigenic polypeptides (e.g., antigenic polypeptides described herein include, for example, CP1, SPP2, etc. as described herein). In some embodiments, the carrier protein is a toxoid or a glycoprotein derived from a pathogen. Other exemplary carrier proteins include, but are not limited to, PspA as described in WO 19 / 167008; CRM197 as described in WO 14 / 92378; variants of CRM197 disclosed in WO 22 / 178015, such as eCRM197; diphtheria toxoid; variants of diphtheria toxoid as described in WO 18 / 156465; tetanus toxoid as described in WO 19 / 152921; extraprotein A and protein D as described in US5858677, and combinations thereof. In some embodiments, the carrier protein is CRM197.

[0220] In some embodiments, the fusion protein described herein may include a carrier protein.

[0221] Exemplary methods for isolating and purifying polysaccharides

[0222] In some embodiments, this disclosure provides methods for purifying one or more polysaccharides described herein from one or more bacterial cell components. In some embodiments, the method includes purifying capsular polysaccharides from one or more bacterial cell components.

[0223] In some embodiments, the bacteria are Gram-negative. In some embodiments, the bacteria are Gram-positive. In some embodiments, the bacteria are... Streptococcus pneumoniae .

[0224] In some embodiments, the cellular component includes proteins. In some embodiments, the cellular proteins include nucleic acids. In some embodiments, the cellular component includes lipids. In some embodiments, the cellular component includes polysaccharides. In some embodiments, the cellular component is part of a lysate.

[0225] In some embodiments, the polysaccharide purification process incorporates a series of steps including ethanol precipitation, washing of the crude polysaccharide formulation with ethanol, diethyl ether, and / or acetone, and drying under vacuum to obtain the purified product. In some embodiments, a phenol extraction step is incorporated for polysaccharide purification. In some embodiments, the purification process employs a CTAB (hexadecyltrimethylammonium bromide) precipitation step in addition to the ethanol and phenol precipitation steps.

[0226] Exemplary methods for biotinylating polysaccharides

[0227] In some embodiments, this disclosure provides a method for biotinylating one or more polysaccharides described herein. In some embodiments, the method includes reacting a purified polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to activate hydroxyl groups in the polysaccharide, followed by the addition of amine PEG biotin under conditions that induce covalent bonding between biotin and the polysaccharide. In some embodiments, the desired level of biotinylation is achieved by varying the ratio of CDAP to the polysaccharide. In some embodiments, the biotinylated polysaccharide is purified by filtration to remove process residues such as unreacted biotin, dimethylaminopyridinium, acetonitrile, cyanide, and unreacted glycine. In some embodiments, the biotinylation level of the polysaccharide described herein is optimized to reduce the amount of biotin available after MAPS complexation.

[0228] Immunogenic complexes

[0229] In some embodiments, the immunogenic agents described herein are or comprise immunogenic complexes. In some embodiments, the immunogenic complex is or comprises one or more polypeptides described herein that are non-covalently complexed with a polysaccharide described herein (e.g., a purified capsular polysaccharide of Formula I). ​​In some embodiments, one or more polypeptides are complexed via an affinity interaction with the polysaccharide (e.g., a purified capsular polysaccharide of Formula I). ​​In some embodiments, the immunogenic agents of this disclosure comprise one or more polypeptides non-covalently complexed with a polysaccharide (e.g., a purified capsular polysaccharide of Formula I) using one or more affinity molecule pairs, each affinity molecule pair comprising a first affinity molecule and a second affinity molecule complementary to the first affinity molecule (“complementary affinity molecule”). After the first affinity molecule binds to the complementary affinity molecule, one or more polypeptides non-covalently complex with the purified capsular polysaccharide of Formula I.

[0230] In some embodiments, the polypeptide of the immunogenic complex is a fusion protein comprising a biotin-binding moiety and at least one polypeptide antigen. In some embodiments, biotinylated purified capsular polysaccharide non-covalently associates with the biotin-binding moiety of the fusion protein.

[0231] In some embodiments, the immunogenic complexes described herein comprise one or more polypeptides (e.g., antigenic polypeptides) that are complexed with purified capsular polysaccharides via affinity interactions. In some embodiments, the immunogenic agents of this disclosure comprise one or more polypeptides (e.g., antigenic polypeptides) non-covalently complexed with purified capsular polysaccharides using an affinity molecule / complementary affinity molecule pair. In some embodiments, the immunogenic agents of this disclosure comprise one or more polypeptides (e.g., antigenic polypeptides) non-covalently complexed with purified capsular polysaccharides using one or more affinity molecule / complementary affinity molecule pairs. In some embodiments, each affinity molecule / complementary affinity molecule pair is identical, e.g., a biotin / biotin-binding moiety pair. In some embodiments, the immunogenic agent comprises (i) a purified capsular polysaccharide (e.g., purified capsular polysaccharide of formula I) associated with a first affinity molecule described herein (e.g., in some embodiments, by chemical conjugation) and (ii) a fusion protein comprising the complementary affinity molecule described herein and one or more polypeptides (e.g., antigenic polypeptides). In some embodiments, the immunogenic agent comprises (i) a purified capsular polysaccharide (e.g., the purified capsular polysaccharide of Formula I) associated with (e.g., in some embodiments, by chemical conjugation) a plurality of first affinity molecules described herein and (ii) a fusion protein comprising a complementary affinity molecule described herein and one or more polypeptides (e.g., an antigenic polypeptide). Upon binding of the first affinity molecule and the complementary affinity molecule, one or more polypeptides (e.g., affinity polypeptides) non-covalently complex with the purified capsular polysaccharide (e.g., the purified capsular polysaccharide of Formula I).

[0232] In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of the following: biotin / biotin-binding moieties, antibodies / antigens, enzymes / substrates, receptors / ligands, metal / metal-binding proteins, carbohydrate / carbohydrate-binding proteins, lipid / lipid-binding proteins, and His-tagged / His-tagged binding molecules.

[0233] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is a portion (e.g., a biotin-binding protein) or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding portion is or comprises rhizobiacin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrAvidin, or CaptAvidin. TMOr its biotin-binding domain or biotin-binding fragment, or a combination thereof. In some embodiments, the biotin-binding moiety is a dimer, for example, a non-covalent dimer. In some embodiments, the biotin-binding moiety is a rhizobium or a variant thereof, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 1, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding moiety is or comprises a polypeptide containing at least 80% (including, for example, at least 90%, at least 95%, at least 98%, at least 99%, and 100%) the same amino acid sequence as SEQ ID NO: 1, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding portion is or comprises a polypeptide containing at least 80% (including, for example, at least 90%, at least 95%, at least 98%, at least 99%, and 100%) of the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 3, or its biotin-binding domain or biotin-binding fragment.

[0234] In some embodiments, one or more antigenic peptides are covalently linked (e.g., fused) to a complementary affinity molecule described herein. In some embodiments, the fusion protein comprises one or more antigenic peptides and a complementary affinity molecule disclosed herein. In some embodiments, the complementary affinity molecule is or comprises a biotin-binding moiety. In some embodiments, the biotin-binding moiety is a dimer, e.g., a non-covalent dimer. In some embodiments, the biotin-binding moiety comprises rhizobialin or its biotin-binding moiety.

[0235] In some embodiments, the immunogenic agents of this disclosure are immunogenic complexes as described in WO 2018 / 237221, WO 2020 / 056202, PCT / US2022 / 043156, or PCT / US2022 / 042964. For the purposes described herein, the entire contents of each of the foregoing references are incorporated herein by reference.

[0236] In some embodiments, the immunogenic agents described herein are or are based on multiple antigen presentation system (MAPS) complexes. Aspects of the MAPS platform have previously been described in WO2012 / 155007 and WO2020 / 056202, the entire contents of which are incorporated herein by reference.

[0237] Immunogenic conjugates

[0238] In some embodiments, the immunogenic agents described herein are or comprise immunogenic conjugates. In some embodiments, the immunogenic conjugates are or comprise one or more polypeptides described herein covalently conjugated to the polysaccharide described herein (e.g., purified capsular polysaccharide of Formula I).

[0239] In some embodiments, the polypeptide of the immunogenic conjugate is or comprises the antigen polypeptide described herein. In some embodiments, the polypeptide of the immunogenic conjugate is or comprises the carrier protein described herein. In some embodiments, the polypeptide of the immunogenic conjugate is or comprises the fusion protein described herein.

[0240] Manufacturing of immunogenic complexes and immunogenic conjugates

[0241] This disclosure includes methods for manufacturing the immunogenic complexes described herein. In some embodiments, the method of manufacturing the immunogenic complex includes compounding at least one biotinylated polymer (e.g., a biotinylated polysaccharide (e.g., the biotinylated polysaccharide described herein)) with at least one biotin-binding fusion protein described herein.

[0242] In some embodiments, this disclosure includes methods for manufacturing the immunogenic conjugates described herein. In some embodiments, the method for manufacturing the immunogenic conjugates includes conjugating at least one polymer described herein (e.g., a polysaccharide (e.g., an antigenic polysaccharide)) with at least one polypeptide described herein (e.g., an antigenic polypeptide).

[0243] In some embodiments, the average (e.g., mean) protein (e.g., antigen protein) to polymer (e.g., polysaccharide) ratio of the plurality of immunogenic compositions or immunogenic conjugates is about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the plurality of immunogenic complexes or immunogenic conjugates is about 1:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the plurality of immunogenic complexes or immunogenic conjugates is about 2:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 3:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 4:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 5:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 6:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 7:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 8:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 9:1 (w / w). In some embodiments, the average protein to polymer (e.g., polysaccharide) ratio of the multiple immunogenic complexes or immunogenic conjugates is about 10:1 (w / w). The immunogenic complexes and vaccines of this disclosure may comprise mixtures of immunogenic complexes or immunogenic conjugates with different average protein to polymer (e.g., polysaccharide) ratios. In some embodiments, the average protein to polymer ratio in the multiple immunogenic complexes is selected to enhance the immunogenic potential (carrier or presentation function) of the polymer and / or to elicit protection against or inhibit pathogens through protein-specific immune responses. The immunogenic compositions (e.g., vaccines) of this disclosure may comprise mixtures of immunogenic complexes and / or immunogenic conjugates with different average protein to polymer ratios.

[0244] In some embodiments, the immunogenic composition (e.g., a vaccine) comprises multiple immunogenic complexes comprising the fusion protein and capsular polysaccharide described herein. In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the multiple immunogenic complexes is about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1 (w / w). In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the multiple immunogenic complexes is about 1:1 (w / w). In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the multiple immunogenic complexes is about 2:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 3:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 5:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 6:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 8:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 9:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the multiple immunogenic complexes is about 10:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in a variety of immunogenic complexes is selected to enhance the immunogenic potential (carrier or presentation function) of the polysaccharide and / or to induce protection or inhibition against pneumococcal colonization of any pneumococcus (independent of polysaccharide serotype) through a protein-specific immune response. The immunogenic compositions of this disclosure (e.g., vaccines) may comprise a mixture of immunogenic complexes with different average fusion protein to capsular polysaccharide ratios.

[0245] Characterization of immunogenic compositions

[0246] In some embodiments, this disclosure provides a method for characterizing an immunogenic composition comprising purified capsular polysaccharide having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, and the method comprises the following steps: characterizing a plurality of units of formula I in the purified capsular polysaccharide, wherein R is –C(O)-CH3; and comparing the number of units of formula I (where R is –C(O)-CH3) with a reference standard.

[0247] The immunogenic compositions described herein can be characterized using methods known to those skilled in the art. For example, in some embodiments, the immunogenic composition is characterized by determining the structure of a purified capsular polysaccharide (e.g., a purified capsular polysaccharide of Formula I). ​​In some embodiments, the immunogenic composition is characterized by evaluating the weight-average molecular weight of the purified capsular polysaccharide in the immunogenic composition. In some embodiments, the immunogenic composition is characterized by evaluating the percentage of O-acetylated groups (i.e., where R is –C(O)-CH3) in the purified capsular polysaccharide of Formula I in the immunogenic composition. In some embodiments, the purified capsular polysaccharide is characterized by nuclear magnetic resonance (NMR). In some embodiments, the immunogenic composition is characterized by comparison with a reference standard, wherein the reference standard is a composition comprising a purified capsular polysaccharide (e.g., a purified capsular polysaccharide of Formula I) in a known form and amount.

[0248] In some embodiments, this disclosure provides a method for characterizing an immunogenic composition comprising purified capsular polysaccharide having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, wherein the method includes the steps of: quantifying the unit percentage of the purified capsular polysaccharide of Formula I; and comparing the unit percentage of Formula I in the purified capsular polysaccharide with a reference standard.

[0249] In some embodiments, this disclosure provides a method for selecting the acetylation level of purified capsular polysaccharides of an immunogenic composition, the method comprising the steps of administering to each of a variety of animals one of a plurality of compositions comprising purified capsular polysaccharides having repeating units of Formula I. I Each R is independently selected from H and –C(O)-CH3, wherein in each of the multiple compositions, R is –C(O)-CH3, which is a unique percentage of the total number of units of Formula I; determining the level of immune response to the purified capsular polysaccharide; comparing the level of the immune response to a reference level; and if the determined immune response is higher than the reference level, selecting a purified capsular polysaccharide with a given acetylation level and incorporating it into the immunogenic composition.

[0250] Immunogenic compositions (e.g., vaccines)

[0251] Another aspect of this disclosure provides immunogenic compositions comprising one or more of the immunogenic agents described herein. For example, an immunogenic composition (e.g., a vaccine composition) may comprise one or more of the immunogenic agents described herein.

[0252] In some embodiments, this disclosure provides an immunogenic composition comprising one or more of the antigenic polysaccharides described herein. In some embodiments, this immunogenic composition comprises: (i) a purified capsular polysaccharide described herein (e.g., a purified capsular polysaccharide of serotype 38), and (ii) one or more other antigenic polysaccharides described herein (e.g., capsular polysaccharides). In some embodiments, this immunogenic composition does not contain polypeptides (e.g., antigenic polypeptides and / or carrier proteins described herein). In some embodiments, one or more antigenic polysaccharides of this immunogenic composition are not associated with polypeptides (e.g., antigenic polypeptides and / or carrier proteins described herein) (e.g., covalently or non-covalently). In some embodiments, this immunogenic composition is a pneumococcal polysaccharide vaccine (PPV).

[0253] In some embodiments, this disclosure provides an immunogenic composition comprising (i) a purified capsular polysaccharide and (ii) a polypeptide, wherein the purified capsular polysaccharide comprises repeating units of formula I: I Each R is independently selected from H and –C(O)-CH3. In some embodiments, R is H. In some embodiments, R is –C(O)-CH3.

[0254] In some embodiments, the immunogenic composition comprises: (i) a purified capsular polysaccharide of Formula I and (ii) one or more polypeptides described herein (e.g., antigenic polypeptides and / or carrier proteins described herein). In some embodiments, the purified capsular polysaccharide of Formula I is associated (e.g., covalently or non-covalently) with the polypeptide (e.g., antigenic polypeptides and / or carrier proteins described herein). In some embodiments, the polypeptide is a carrier protein, for example selected from: CRM197, tetanus toxoid, extracellular protein A, PspA, diphtheria toxoid, and protein D. In some embodiments, the carrier protein is CRM197. In some embodiments, the polypeptide is or comprises a pneumococcal polypeptide antigen, which may be selected from: pneumococcal hemolysin polypeptide antigen, SP0435 polypeptide antigen, SP0785 polypeptide antigen, and SP1500 polypeptide antigen.

[0255] In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is from about 10:1 to about 1:10. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is from about 20:1 to about 1:2. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is from about 5:1 to about 1:5. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is from about 3:1 to about 1:3. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is from about 5:1 to about 1:1. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is about 3:1, 2:1, 1:1, 1:2, or 1:3. In some embodiments, the weight ratio of the peptide to the purified capsular polysaccharide in the immunogenic composition described herein is about 3:1.

[0256] In some embodiments, the immunogenic composition may comprise a group of one type of immunogenic complex or immunogenic conjugate, wherein all immunogenic complexes or immunogenic conjugates comprise the same polypeptide (e.g., antigenic polypeptide) and a purified capsular polysaccharide of Formula I. In some embodiments, such a composition may comprise multiple immunogenic agents of one type described herein. For example, the immunogenic composition may comprise a group of one type of immunogenic agents, wherein all immunogenic compositions comprise the same polypeptide (e.g., antigenic polypeptide) and the same polymer (e.g., antigenic polysaccharide). Alternatively, such a composition may comprise multiple immunogenic complexes or immunogenic conjugates of more than one type described herein. For example, the immunogenic composition may comprise a group of different types of immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition may comprise a group of first-type immunogenic complexes or immunogenic conjugates and a group of second-type immunogenic complexes or immunogenic conjugates, wherein the first-type and second-type immunogenic complexes or immunogenic conjugates have different polypeptides (e.g., antigenic polypeptides) and / or different polymers (e.g., antigenic polysaccharides). In some embodiments, the immunogenic composition may comprise a group of first-type immunogenic complexes or immunogenic conjugates and a group of second-type immunogenic complexes or immunogenic conjugates, wherein the first-type and second-type immunogenic complexes or immunogenic conjugates comprise the same polypeptide (e.g., antigenic polypeptide) and different polymers (e.g., antigenic polysaccharides (e.g., polysaccharides of different serotypes)).

[0257] In some embodiments, the immunogenic composition is a monovalent immunogenic composition. In some embodiments, the immunogenic composition is a polyvalent or multivalent immunogenic composition. In some embodiments, the immunogenic composition is a monovariate immunogenic composition comprising one or more antigens from a single strain or variant of a pathogen. In some embodiments, the immunogenic composition is a multivariate immunogenic composition comprising one or more antigens from more than one strain or variant of a pathogen. In some embodiments, the valence of the immunogenic composition refers to the number of types of immunogenic complexes or immunogenic conjugates present in the immunogenic composition. The valence of the immunogenic composition described herein is not limited to the total antigen present in the immunogenic composition, nor is it limited to the number of pathogen strains from which an immune protective response can be induced by application of the immunogenic composition.

[0258] In some embodiments, the immunogenic composition comprises 1-60 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-50 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-45 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-40 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-35 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-34 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-33 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-32, 1-31, or 1-30 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-30 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-30 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-24 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-15 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-9 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises 1-5 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition is a polyvalent composition.

[0259] In some embodiments, the immunogenic composition comprises at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 immunogenic complexes or immunogenic conjugates. In some embodiments, the immunogenic composition comprises at least 30 (including, for example, at least 31, at least 32, at least 33, or at least 34) of the immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises 30-40 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises 30-35 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises 25-50 or 25-40 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises no more than 60 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises no more than 50 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises no more than 45 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises no more than 40 immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises no more than 35 immunogenic complexes or immunogenic conjugates described herein.

[0260] In some embodiments, the immunogenic composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 of the immunogenic complexes or immunogenic conjugates described herein. In some embodiments, the immunogenic composition comprises an immunogenic complex or immunogenic conjugate containing purified capsular polysaccharide (e.g., of Formula I) of serotype 38 described herein. In some embodiments, the immunogenic composition comprises an immunogenic complex or immunogenic conjugate containing purified capsular polysaccharide of serotype 38 as described herein (e.g., of formula I), and further comprises an immunogenic complex or immunogenic conjugate containing one or more Streptococcus pneumoniae bacteria. Streptococcus pneumoniae The polysaccharide comprises each of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B. In some embodiments, the immunogenic composition comprises an immunogenic complex or immunogenic conjugate comprising purified capsular polysaccharide of serotype 38 described herein (e.g., of formula I), and further comprises an immunogenic complex or immunogenic conjugate comprising each of the following Streptococcus pneumoniae strains (…). Streptococcus pneumoniae Serum-type polysaccharides: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B.

[0261] In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the weight of the polymer (e.g., polysaccharide) in the composition from each immunogenic complex or immunogenic conjugate is substantially the same, for example, present at a w / w ratio of about 1:1. In some embodiments, the weight of polymer (e.g., polysaccharide) contributed by each immunogenic complex or immunogenic conjugate to the composition is about 0.20 μg, about 0.25 μg, about 0.5 μg, about 1 μg, about 1.5 μg, about 2 μg, about 2.5 μg, about 3 μg, about 3.5 μg, about 4 μg, about 4.5 μg, about 5 μg, about 5.5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, or about 12 μg. In some embodiments, the weight of polymer (e.g., polysaccharide) contributed by each immunogenic complex or immunogenic conjugate in the composition is greater than 12 μg, for example, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg or higher.

[0262] In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the weight of the polymer (e.g., polysaccharide) in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is different, for example, present in a w / w ratio that is not about 1:1. In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the weight of the polymer (e.g., polysaccharide) in the immunogenic conjugate contributed by the first immunogenic complex or immunogenic conjugate and the second immunogenic complex or immunogenic conjugate is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes or immunogenic conjugates such that the weight of the polymer (e.g., polysaccharide) in the immunogenic composition contributed by the immunogenic complex or immunogenic conjugate ranges from about 0.20 μg to about 6 μg. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes or immunogenic conjugates such that the weight range of the polymer (e.g., polysaccharide) in the immunogenic composition contributed by the immunogenic complex or immunogenic conjugate is from about 0.20 μg to about 12 μg. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes or immunogenic conjugates such that the weight range of the polymer (e.g., polysaccharide) in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is from about 0.20 μg to about 20 μg. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes or immunogenic conjugates such that the weight range of the polymer (e.g., polysaccharide) in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is from about 0.20 μg to about 40 μg.

[0263] In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the combined weight of the polymer (e.g., polysaccharide) and peptide in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is substantially the same, for example, a w / w protein:polysaccharide ratio of about 1:1. In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the combined weight of the polymer (e.g., polysaccharide) and peptide in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is a w / w protein:polysaccharide ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0264] In some embodiments, the combined weight of the polymer (e.g., polysaccharide) and peptide in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is about 0.20 μg, about 0.40 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 16 μg, about 18 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, or about 110 μg.

[0265] In some embodiments, the immunogenic composition comprises two or more immunogenic complexes or immunogenic conjugates in amounts such that the combined weight of the polymer (e.g., polysaccharide) and peptide in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate is different, for example, present in a w / w protein:polysaccharide ratio that is not about 1:1, for example, the protein:polysaccharide ratio is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes or immunogenic conjugates such that the combined weight of the polymer (e.g., polysaccharide) and peptide in the immunogenic composition contributed by each immunogenic complex or immunogenic conjugate ranges from about 0.4 μg to about 110 μg.

[0266] In some embodiments, the immunogenic compositions described herein contain an adjuvant.

[0267] In some embodiments, the immunogenic compositions described herein are vaccine compositions.

[0268] In some embodiments, the immunogenic composition is the immunogenic composition described in WO 2020 / 056202, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the immunogenic composition is the immunogenic composition described in International Application PCT / US2022 / 043156, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0269] Pharmaceutical Composition

[0270] In some embodiments, the compositions described herein (e.g., immunogenic compositions) are formulated into pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0271] The optimal amount of a component used in a particular pharmaceutical composition can be determined through standard studies involving observation of an appropriate immune response in subjects. Following the initial vaccination, subjects may receive one or more booster immunizations spaced sufficiently apart in time.

[0272] The immunogenic agents and / or formulations described herein may be formulated in unit dosage forms for ease of administration and dosage uniformity. Therapeuticly effective dose levels specific to any particular subject or organism may depend on a number of factors including: the severity or risk of infection; the activity of the particular pharmaceutical composition used; other characteristics of the particular pharmaceutical composition used; the subject's age, weight, general health condition, sex, diet, pharmacokinetic status, timing of administration (e.g., with respect to other subject activities such as eating, sleeping, receiving other drugs including other pharmaceutical compositions), route of administration, excretion rate of the particular pharmaceutical composition used; pharmaceutical compositions used in combination with or concurrently with the pharmaceutical composition used; and similar factors well known in the medical field.

[0273] Immunogenic agents used according to this disclosure can be formulated into compositions (e.g., pharmaceutical compositions) using known techniques. In some embodiments in which the pharmaceutical composition comprises a vaccine composition, vaccine preparation is generally described in Vaccine Design (Powell and Newman, 1995). For example, an immunogenic amount of the vaccine composition can be formulated with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials.

[0274] Generally, pharmaceutically acceptable carriers include solvents and dispersion media that are compatible with drug administration. For example, materials that can serve as pharmaceutically acceptable carriers, according to the formulation engineer's judgment (Martin, 1975), include, but are not limited to, sugars such as lactose, glucose, dextrose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragali; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; polyols such as glycerol, propylene glycol, and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as preservatives and antioxidants, which may also be present in the composition.

[0275] Pharmaceutical compositions can be formulated by combining one or more of the immunogenic agents disclosed herein with a carrier and / or other optional components via any available means, including, for example, conventional mixing, granulation, dissolution, lyophilization or similar processes.

[0276] The pharmaceutical composition applicable to the provided methods can be lyophilized until it is about to be used, during which time it is temporarily reconstituted with a diluent. In some embodiments, the pharmaceutical composition or its components are lyophilized in the presence of one or more other components (e.g., adjuvants) and temporarily reconstituted with a saline solution. Alternatively, individual components or sets of components can be lyophilized and / or stored separately (e.g., in a vaccine kit), the components can be reconstituted, and then mixed or administered separately to the subject prior to use.

[0277] Lyophilization can produce more stable compositions (e.g., by preventing or reducing the degradation of polysaccharide antigens). Lyophilization of pharmaceutical compositions or components thereof is well known in the art. Typically, liquid pharmaceutical compositions or components thereof are lyophilized in the presence of an anti-caking agent (such as, for example, sugar, such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10-200 mg / ml. Lyophilization typically occurs in a series of steps, for example, cycling starting at -69°C, gradually adjusting to -24°C over 3 hours, then maintaining this temperature for 18 hours, followed by gradually adjusting to -16°C over 1 hour, then maintaining this temperature for 6 hours, followed by gradually adjusting to +34°C over 3 hours, and finally maintaining this temperature for more than 9 hours.

[0278] In some embodiments, the pharmaceutical composition is a liquid. In some embodiments, the liquid is a reconstituted lyophylate. In some embodiments, the pH of the pharmaceutical composition is about 5, about 6, about 7, or about 8. In some embodiments, the pH of the pharmaceutical composition is between about 5 and about 7.5. In some embodiments, the pH of the pharmaceutical composition is between 5 and 7.5. In some embodiments, the pH of the pharmaceutical composition is between about 5.3 and about 6.3. In some embodiments, the pH of the pharmaceutical composition is between 5.3 and 6.3. In some embodiments, the pH of the pharmaceutical composition is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0279] The pharmaceutical compositions or components thereof used according to this disclosure may be incorporated into liposomes, cochleates, biodegradable polymers (such as polylactide, polyglycolic acid, and polylactide-co-glycolic acid) or immunostimulatory complexes (ISCOM).

[0280] In some cases, it may be necessary to prolong the action of the pharmaceutical composition used according to this disclosure, for example, by slowing the absorption of one or more pharmaceutical composition components. Such absorption delay can be achieved, for example, by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the product then depends on its dissolution rate, which in turn may depend on its size and form. Alternatively, delayed absorption can be achieved by dissolving or suspending one or more pharmaceutical composition components in an oil medium. An injectable depot form can also be used to delay absorption. Such a depot form can be prepared by forming a biodegradable polymer network from a microcapsule matrix of one or more pharmaceutical composition components. The release rate can be controlled depending on the ratio of polymer to pharmaceutical composition component and the properties of the one or more specific polymers used.

[0281] Examples of biodegradable polymers that may be used according to this disclosure include, for example, poly(orthoester) and poly(anhydride). A particular exemplary polymer is polylactide-polyglycolic acid.

[0282] Depot injectable formulations can also be prepared by encapsulating the product in liposomes or microemulsions that are compatible with body tissues.

[0283] Polymer delivery systems can also be used in non-reservoir formulations, including, for example, oral formulations. For instance, biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used in oral formulations. Polysaccharide antigens or conjugates can be formulated with such polymers, for example, to prepare particles, microparticles, extrudates, solid dispersions, mixtures, or other combinations to facilitate the preparation of usable formulations, such as oral formulations.

[0284] The pharmaceutical compositions used according to this disclosure include immunogenic compositions and may additionally include one or more additional active agents (i.e., agents that exert biological effects—non-inert components). For example, one or more adjuvants are often included in the preparation of pharmaceutical compositions. It should be understood that such additional agents may be formulated together with one or more other pharmaceutical composition components, or may be held separately and combined at or near the time of administration. In some embodiments, such additional components may be administered separately from some or all of the other pharmaceutical composition components within an appropriate time window for achieving the relevant effect.

[0285] adjuvant

[0286] The immunogenic compositions (e.g., vaccine compositions) and pharmaceutical compositions described herein may contain adjuvants. Generally, adjuvants are agents that enhance the immune response against an antigen. Adjuvants can be broadly classified into two categories based on their primary mechanism of action: vaccine delivery systems and immunostimulatory adjuvants (see, for example, Singh et al., 2003). In most immunogenic compositions or pharmaceutical compositions, the adjuvant signals the immune system to generate a response against the antigen, and the antigen is required to drive the specificity of the response against the pathogen. Vaccine delivery systems are often particulate formulations such as emulsions, microparticles, immunostimulatory complexes (ISCOMs), nanoparticles (which may be, for example, particles and / or matrices), and liposomes. In contrast, immunostimulatory adjuvants are sometimes derived from or originate from pathogens and may represent pathogen-associated molecular patterns (PAMPs) that activate cells of the innate immune system, such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), or CpG-containing DNA.

[0287] Alternatively, adjuvants can be classified as organic adjuvants and inorganic adjuvants. Inorganic adjuvants include aluminum-based adjuvants (e.g., alum salts) commonly used in human immunogenic compositions and pharmaceutical compositions, such as aluminum phosphate, amorphous aluminum sulfate hydroxyphosphate, and aluminum hydroxide. Organic adjuvants comprise organic molecules that include macromolecules. Non-limiting examples of organic adjuvants include cholera toxin / toxin-like substances, other enterotoxins / toxin-like substances or unstable toxins / toxin-like substances from Gram-negative bacteria, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma-interferon), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0288] Adjuvants can also be classified according to the response they induce. In some embodiments, adjuvants induce the generation, proliferation, or activation of TH1 or TH2 cells. In other embodiments, adjuvants induce the generation, proliferation, or activation of B cells. In still other embodiments, adjuvants induce the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, adjuvants activate more than one type of cell.

[0289] In some implementations, the adjuvant induces the generation, proliferation, or activation of TH17 cells. The adjuvant can promote CD4+ activation. + T cells or CD8 + T cells secrete IL-17. In some embodiments, the adjuvant that induces the generation, proliferation, or activation of TH17 cells is an adjuvant that produces an experimental sample to control ratio of at least 2 and, in some cases, 10 times in the following assay. In the assay, the experimenter compares the IL-17 levels secreted by two cell populations: (1) cells from animals immunized with an adjuvant and a polypeptide known to induce the generation, proliferation, or activation of TH17, and (2) cells from animals treated with an adjuvant and an unrelated (control) polypeptide. The adjuvant that induces the generation, proliferation, or activation of TH17 cells can cause the cells in population (1) to produce more than 2 times or more than 10 times more IL-17 than the cells in population (2). IL-17 can be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and unstable toxins (by enterotoxicity) E. coli(Or generated by ETEC) activates the TH17 reaction. Therefore, in some embodiments, the adjuvant is a toxin or a toxin-like substance. Mutant derivatives of unstable toxins (toxin-like substances) that have activity as adjuvants but significantly lower toxicity can also be used. Exemplary detoxified mutant derivatives of unstable toxins include mutants lacking ADP-ribosyltransferase activity. Specific detoxified mutant derivatives of unstable toxins include LTK7 (Douce et al., 1995) and LTK63 (Williams et al., 2004), LT-G192 (Douce et al., 1999), and LTR72 (Giuliani et al., 1998).

[0290] In some embodiments, the adjuvant comprises VLPs (virus-like particles). One such adjuvant platform, the alphavirus replicon, uses alphavirus to induce activation of TH17 cells and is produced by Alphavax. In some embodiments of the alphavirus replicon system, the alphavirus may be engineered to express a target antigen, a target cytokine (e.g., IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in helper cell lines. More detailed information can be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, an immunogenic composition (e.g., a vaccine) is administered to a subject in combination with a nucleic acid encoding a cytokine.

[0291] Certain classes of adjuvants activate toll-like receptors (TLRs) to activate the TH17 response. TLRs are well-known proteins that can be present on leukocyte membranes and recognize foreign antigens, including microbial antigens. Administration of a known TLR ligand along with a target antigen (e.g., in fusion protein form) can promote the development of an immune response specific to the target antigen. An exemplary adjuvant for activating TLRs comprises monophosphoryl lipid A (MPL). Traditionally, adjuvants derived from sources such as… S. minnesotaMPL is obtained from detoxified lipopolysaccharide (LPS) endotoxins derived from Gram-negative bacteria. Specifically, sequential acid and alkali hydrolysis of LPS yields the immunologically active lipid A moiety (which is MPL), lacking all but one of the glycosides present in LPS. Several synthetic TLR agonists, particularly TLR-4 agonists, are disclosed in Evans et al., 2003. Similar to MPL adjuvants, these synthetic compounds activate the innate immune system via TLRs. Another type of TLR agonist is a synthetic phospholipid dimer, such as E6020 (Ishizaka et al., 2007). Various TLR agonists, including TLR-4 agonists, have been manufactured and / or marketed by companies such as the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti PolarLipids, Inc., and Sigma Aldrich. Another exemplary adjuvant for TLR activation comprises a mixture of MPL, trehalose dicoynomycolate (TDM), and bis(octadecyl dimethyl ammonium bromide) (DDA). Another TLR activating adjuvant is R848 (resiquimod).

[0292] In some embodiments, the adjuvant is or comprises saponin. Typically, saponins are triterpenoid glycosides, such as those isolated from the bark of the soapberry tree (Quillaja saponaria). Extracts of saponins from biological sources can be further fractionated (e.g., by chromatography) to separate fractions of the extract that have optimal adjuvant activity and acceptable toxicity. Typical fractions of the soapberry tree extract used as adjuvants are referred to as fractions A and C.

[0293] In some implementations, combinations of adjuvants are used. Three exemplary adjuvant combinations are MPL and alum, E6020 and alum, and MPL and ISCOM.

[0294] Adjuvants can bind covalently or nonvalently to antigens. In some embodiments, adjuvants may contain proteins that induce an inflammatory response through activation of antigen-presenting cells (APCs). In some embodiments, one or more of these proteins may be fused to a selected antigen in a recombinant manner such that the resulting fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately enhances antigen presentation to T cells and the initiation of T cell responses (e.g., see Wu et al., 2012).

[0295] In some embodiments, the immunogenic agents described herein are formulated and / or administered in combination with adjuvants. In some embodiments, such adjuvants are or comprise aluminum-based adjuvants (e.g., aluminum salts); see, for example, Danielsson and Eriksson, “Aluminum adjuvants in vaccines – a way to modulate the immune response”. Semin Cell Dev Biol , 115:3-9 (2021) and HogenEsch et al., "Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want", NPJ Vaccines , 3:51 (2018), the contents of which are incorporated herein by reference in their entirety for the purposes described herein. Examples of aluminum-based adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate (alum), aluminum hydroxide phosphate, aluminum hydroxyphosphate sulfate, and combinations thereof. In some embodiments, the aluminum-based adjuvant comprises aluminum phosphate. In some embodiments, the aluminum-based adjuvant is aluminum phosphate.

[0296] In some embodiments, the same adjuvant or adjuvant mixture is present in each dose of the immunogenic composition or pharmaceutical composition. However, optionally, the adjuvant may be administered with the first dose of the immunogenic composition or pharmaceutical composition, but not with subsequent doses (i.e., booster injections). Alternatively, a strong adjuvant may be administered with the first dose of the immunogenic composition or pharmaceutical composition, while a weaker adjuvant or a lower dose of a strong adjuvant may be administered with subsequent doses. The adjuvant may be administered before, simultaneously with, or after the administration of the antigen to the subject (sometimes within 1, 2, 6, or 12 hours, sometimes within 1, 2, or 5 days). Certain adjuvants are suitable for human subjects, non-human animals, or both.

[0297] The immunogenic compositions and pharmaceutical compositions used according to this disclosure may contain other antimicrobial or anti-inflammatory therapies, or may be administered concurrently with other antimicrobial or anti-inflammatory therapies. For example, such immunogenic compositions and pharmaceutical compositions may contain one or more agents that kill or inhibit pathogen growth, or may be administered concurrently with one or more agents that kill or inhibit pathogen growth. Such agents include, for example, remdesivir, lopinavir and / or ritonavir (e.g., Kaletra), oseltamivir (Tamiflu), favipiravir, umifenvironvir, galixivir, dexamethasone, colchicine, convalescent plasma, monoclonal antibodies (e.g., one or more of bamlanivimab, LY-CoV016, etesevimab, casirivimab, indevimab, sarilumab, tocilizumab), IL-6 inhibitors, kinase inhibitors, interferon, penicillin, vancomycin, erythromycin, azithromycin, clarithromycin, cefotaxime, ceftriaxone, levofloxacin, and gatifloxacin.

[0298] Alternatively or otherwise, the immunogenic compositions and pharmaceutical compositions used according to this disclosure may comprise one or more other immunogenic compositions, pharmaceutical compositions or therapies, or may be administered together with one or more other immunogenic compositions, pharmaceutical compositions or therapies.

[0299] Additional components and excipients

[0300] In addition to the antigens and adjuvants described above, immunogenic compositions (e.g., vaccine compositions) or pharmaceutical compositions may also contain one or more additional components.

[0301] In some embodiments, the immunogenic composition or pharmaceutical composition may contain one or more stabilizers such as: sugars (such as sucrose, glucose, or fructose), phosphates (such as disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium dihydrogen phosphate), glutamates (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and their alkyl esters), inosine, or sodium borate.

[0302] In some embodiments, the immunogenic composition or pharmaceutical composition comprises one or more buffers, such as histidine, glycine, succinate, Tris, or a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in saline solution such as phosphate-buffered saline (PBS) or distilled water.

[0303] In some embodiments, the immunogenic composition or pharmaceutical composition comprises one or more surfactants, such as, but not limited to, polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20), polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether (TRITON X-100), and a polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with formaldehyde and ethylene oxide (TYLOXAPOL). The surfactant may be an ionic surfactant or a nonionic surfactant.

[0304] In some embodiments, the immunogenic composition or pharmaceutical composition comprises one or more salts, such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

[0305] In some embodiments, the immunogenic composition or pharmaceutical composition includes a preservative. In other embodiments, no preservative is used. Preservatives are most commonly used in multi-dose immunogenic compositions (e.g., vaccines) or pharmaceutical composition vials, and are less frequently required in single-dose immunogenic compositions (e.g., vaccines) or pharmaceutical composition vials. In some embodiments, the preservative is 2-phenoxyethanol, thimerosal, methylparaben and propylparaben, benzyl alcohol, and / or sorbic acid.

[0306] Use of immunogenic compositions and pharmaceutical compositions

[0307] In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions comprising one or more antigenic polysaccharides described herein are characterized by an increase, as measured by ELISA and / or by a functional antibody assay, in one or more of the opsonization potential or immune response against one or more antigenic polysaccharides relative to a predetermined level. In some embodiments, as measured by ELISA and / or by a functional antibody assay, one or more of the opsonization potential or immune response against one or more antigenic polysaccharides is increased by at least 1, 2, 3, 4, or 5 times relative to a predetermined level. In some embodiments, the predetermined level is a pre-immune level. In some embodiments, one or more polypeptides (e.g., antigenic polypeptides) are carrier proteins against one or more antigenic polysaccharides.

[0308] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more pathogens in the subject at a level higher than that of a composition containing only antigenic polysaccharides. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more pathogens in the subject at a level higher than that of a composition containing only antigenic peptides. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a protective immune response.

[0309] In some embodiments, upon administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more strains (or variants or serotypes) of a pathogen. In some embodiments, the pathogen is a bacterial pathogen. In some embodiments, the bacterial pathogen is... Streptococcus pneumoniae .

[0310] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a response to [the specific target of the immunogenic composition]. Streptococcus pneumoniae The immune response. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against [the target / immune system]. Streptococcus pneumoniae An immune response to one or more serotypes. In some embodiments, this immune response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein comprises one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, this immune response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein does not contain one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, this immune response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniaeThe immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein (i) comprises one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more); and (ii) does not contain one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a response against... Streptococcus pneumoniae A protective immune response against one or more serotypes. In some embodiments, this protective response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein comprises one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, this protective response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein does not contain one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, this protective response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Two or more serotypes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of which the immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein (i) contains one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more); and (ii) does not contain one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0311] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the antibody or B cell response is a memory B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the T cell response is a memory T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including T cells... H 1. T H 2 or T H The immune response may be a CD8+ T cell response, a CD4+ and CD8+ T cell response, or a CD4- / CD8- T cell response. In some embodiments, the immune response is an antibody response or a B cell response and a T cell response. In some embodiments, the immune response is an antibody response or a B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response includes neutralizing antibodies. In some embodiments, the immune response is a memory response.

[0312] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces antibody production against one or more pathogens in the subject at a higher level than a composition containing only an antigenic polysaccharide. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces antibody production against one or more pathogens in the subject at a higher level than a composition containing only a peptide antigen.

[0313] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more pathogens in the subject at a higher level than a composition containing only an antigenic polysaccharide. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more pathogens in the subject at a higher level than a composition containing only a polypeptide antigen. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a protective immune response.

[0314] The compositions described herein can be used for the preventive and / or therapeutic treatment of pathogen infection and / or disease. Therefore, this disclosure provides a method for immunizing a subject suffering from or susceptible to pathogen infection, the method comprising administering an immunogenically effective amount of any of the compositions described herein. The subject receiving immunization may be male or female, and may be an infant, child, adolescent, or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal. In some embodiments, upon administration to the subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a protective immune response against pathogen infection and / or disease.

[0315] In a prophylactic implementation, an immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to a subject to induce an immune response that can, for example, help prevent the establishment of one or more strains (variants) of a pathogen by preventing asymptomatic infection. In some aspects, the method inhibits infection of the pathogen in uninfected subjects. In another aspect, the method can reduce the spread, replication, and / or pathogen load of one or more strains (variants) of the pathogen in already infected subjects.

[0316] In a preventative implementation, administering the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein to a subject to induce an immune response can, for example, help prevent colonization (the first and essential step in disease progression). pneumonia chain cocci The establishment of an immune response. In some embodiments, this immune response can target Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein comprises one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, this immune response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein does not contain one or more polysaccharides present in at least one or more of these serotypes (non-vaccine types, NVT). In some embodiments, this immune response may be against Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniaeThe immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein (i) comprises one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more); and (ii) does not contain one or more polysaccharides present in at least one or more of such serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Therefore, in some aspects, the method inhibits the growth of uncolonized or uninfected subjects. Streptococcus pneumoniae Infection. In another aspect, the method can shorten the duration of colonization in already colonized subjects.

[0317] In therapeutic embodiments, an immunogenic composition (e.g., a vaccine) or pharmaceutical composition may be administered to a subject suffering from a pathogen infection in an amount sufficient to treat the subject. In this context, treating the subject means reducing pathogen symptoms and / or pathogen load and / or sequelae in the subject. In some embodiments, treating the subject means shortening the duration or reducing the intensity of symptoms or sequelae. In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition reduces the transmissibility of one or more strains (variants) of a pathogen from an immunized subject to another subject. In some embodiments, the reduction described above is at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, for example, relative to a control (e.g., a control subject).

[0318] In a therapeutic implementation, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to a subject following infection with a pathogen. The compositions described herein may be administered shortly after pathogen infection, such as before the onset of symptoms or sequelae, or during or after the onset of symptoms or sequelae.

[0319] In some embodiments, the compositions described herein confer protective immunity, thereby allowing for delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, in immunized subjects due to exposure to the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein. In some embodiments, for example, the severity of symptoms or sequelae is reduced by at least 25%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a control. In certain embodiments, immunized subjects may not exhibit symptoms or sequelae after infection with a pathogen (asymptomatic infection), or may not be infected by the pathogen. Protective immunity is generally achieved through one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily attributed to secretory IgA (sIGA) antibodies on the mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. sIGA antibodies are generated following a series of events mediated by antigen-processing cells, B lymphocytes, and T lymphocytes, which induce sIGA production by B lymphocytes in mucosal-lined tissues of the body. Humoral immunity is typically attributed to IgG and IgM antibodies in the serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity involving macrophages and T lymphocytes, as well as other mechanisms involving T cells that are antibody-independent. In particular, cellular immunity can be mediated by T cells... H 1 cell or T H 17. Cell-mediated.

[0320] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more strains (variants) of a pathogen.

[0321] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a response to [the specific target of the immunogenic composition]. Streptococcus pneumoniae The immune response. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against [the target / immune system]. Streptococcus pneumoniae An immune response to one or more serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or more). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against the serotypes contained in such composition. Streptococcus pneumoniaeAll serotypes of the immune response. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against all serotypes. Streptococcus pneumoniae Protective immune response against one or more serotypes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 or more). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a protective immune response against the serotypes contained in such immunogenic composition (e.g., a vaccine). Streptococcus pneumoniae Protective immune response for all serotypes.

[0322] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the antibody or B cell response is a memory B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the T cell response is a memory T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including T cells... H 1. T H 2 or T H The immune response may be a CD8+ T cell response, a CD4+ and CD8+ T cell response, or a CD4- / CD8- T cell response. In some embodiments, the immune response is an antibody response or a B cell response and a T cell response. In some embodiments, the immune response is an antibody response or a B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response includes neutralizing antibodies. In some embodiments, the immune response is a memory response.

[0323] In some embodiments, upon administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an antibody response or B-cell response against one or more pathogens in the subject at a level higher than that of a composition containing only an antigenic polysaccharide. In some embodiments, upon administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an antibody response or B-cell response against one or more pathogens in the subject at a level higher than that of a composition containing only a peptide antigen. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0324] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a T-cell response against one or more pathogens in the subject at a level higher than that of a composition containing only an antigenic polysaccharide. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a T-cell response against one or more pathogens in the subject at a level higher than that of a composition containing only a polypeptide antigen. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0325] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents disease caused by infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents one or more symptoms caused by infection with one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents [the disease / condition] caused by [the disease / condition]. Streptococcus pneumoniae Infectious diseases include one or more of the following: invasive pneumococcal disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis.

[0326] In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein inhibit or reduce the incidence or recurrence rate of infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein inhibit or reduce the incidence or recurrence rate of disease caused by infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein inhibit or reduce the incidence or recurrence rate of one or more symptoms caused by infection with one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits or reduces the effects of [the following]: Streptococcus pneumoniae The incidence of one or more of the following: invasive pneumococcal disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis caused by infection.

[0327] In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein reduce the severity of infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein reduce the severity of illness caused by infection with one or more strains (variants) of a pathogen. In some embodiments, after administration to a subject, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein reduce the severity of one or more symptoms caused by infection with one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein reduces the susceptibility of the immunogenic composition to other substances. Streptococcus pneumoniaeInfection with invasive pneumococcal disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis can cause one or more of these conditions with varying degrees of severity.

[0328] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the transmission of one or more strains (variants) of a pathogen from that subject to another subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits asymptomatic infection of one or more strains (variants) of a pathogen in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the replication of one or more strains (variants) of a pathogen in the subject and / or reduces the pathogen load of one or more strains (variants) of a pathogen in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the colonization of one or more strains (variants) of a pathogen in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the colonization of one or more strains (variants) of pathogens on the mucosal surface of the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the colonization of one or more strains (variants) of pathogens in the subject's nasopharynx.

[0329] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more pathogens in the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a protective immune response against one or more pathogens in the subject at a level higher than that of a control composition. In some embodiments, the level is higher than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks and about 6 weeks old. In some embodiments, the human is between about 6 weeks and about 6 years old. In some embodiments, the human is between about 6 years and about 18 years old. In some embodiments, the human is between about 18 years and about 50 years old. In some embodiments, the human is between about 50 years and about 75 years old. In some embodiments, the human is about 75 years old or older.

[0330] In some embodiments, after administration to a subject, the immunogenicity induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is no less than that induced by administration of a control composition (e.g., at least as effective as it). In some embodiments, after administration to a subject, the immunogenicity induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is no less than that induced by administration of a control composition against one or more pathogens (e.g., at least as effective as it). In some embodiments, immunogenicity is determined by measuring the amount of anti-polysaccharide antibodies against one or more polysaccharides present in the immunogenic composition (e.g., a vaccine) or pharmaceutical composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the person's age is approximately 50 to approximately 75 years old. In other embodiments, the person's age is approximately 75 years old or older.

[0331] In some embodiments, the immunogenicity induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein after administration to a subject is superior to the immunogenicity induced by administration of a control composition. In some embodiments, the immunogenicity induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein after administration to a subject is superior to the immunogenicity induced by administration of a control composition against one or more pathogens. In some embodiments, immunogenicity is determined by measuring the amount of anti-polysaccharide antibodies against one or more polysaccharides present in the immunogenic composition (e.g., a vaccine) or pharmaceutical composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is between about 50 years of age and about 75 years of age. In some implementations, the person's age is approximately 75 years or older.

[0332] In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is no less than (e.g., at least as effective as) the functional antibody titer induced by administration of a control composition. In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is no less than (e.g., at least as effective as) the functional antibody titer induced by administration of a control composition against one or more tumors or pathogens. In some embodiments, the functional antibody titer is measured by a opsonization phagocytosis assay (e.g., an assay as described herein). In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is no less than (e.g., at least as effective as) the functional antibody titer induced by administration of a control composition against one or more pathogens, wherein such immunogenic composition (e.g., a vaccine) or pharmaceutical composition does not contain one or more polysaccharides present in at least one of said pathogens (non-vaccine type, NVT). In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks and about 6 weeks old. In some embodiments, the human is between about 6 weeks and about 6 years old. In some embodiments, the human is between about 6 years and about 18 years old. In some embodiments, the human is between about 18 years and about 50 years old. In some embodiments, the human is between about 50 years and about 75 years old. In some embodiments, the human is about 75 years old or older.

[0333] In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is superior to the functional antibody titer induced by administration of a control composition. In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is superior to the functional antibody titer induced by administration of a control composition against one or more pathogens. In some embodiments, the functional antibody titer is measured by a opsonization phagocytosis assay (e.g., an assay as described herein). In some embodiments, after administration to a subject, the functional antibody titer induced by the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is superior to the functional antibody titer induced by administration of a control composition against one or more pathogens, wherein such immunogenic composition (e.g., a vaccine) or pharmaceutical composition does not contain one or more polysaccharides present in at least one of the pathogens (non-vaccine type, NVT). In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks and about 6 weeks old. In some embodiments, the person's age is between about 6 weeks and about 6 years old. In some embodiments, the person's age is between about 6 years and about 18 years old. In some embodiments, the person's age is between about 18 years and about 50 years old. In some embodiments, the person's age is between about 50 years and about 75 years old. In some embodiments, the person's age is about 75 years or older.

[0334] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an immune response against one or more strains (variants) of a pathogen at a level higher than that of a control composition. In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including T cells... H 1. T H 2 or T HA 17-cell response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or a CD4- / CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the level is higher than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0335] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces an antibody or B-cell response against one or more pathogens in the subject at a level higher than that of a control composition. In some embodiments, the level is higher than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of that of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0336] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein induces a T-cell response against one or more pathogens in the subject at a level higher than that of a control composition. In some embodiments, the level is higher than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of that of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0337] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents disease caused by infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein treats or prevents one or more symptoms caused by infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some implementations, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, the level is higher than that of the control composition by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0338] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits or reduces the incidence or recurrence rate of infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits or reduces the incidence or recurrence rate of disease caused by infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits or reduces the incidence or recurrence rate of one or more symptoms caused by infection with one or more strains (variants) of a pathogen at a level higher than that of a control composition in the subject. In some implementations, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, the level is higher than that of the control composition by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0339] In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein reduces the severity of infection with one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein reduces the severity of illness caused by infection with one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein reduces the severity of one or more symptoms caused by infection with one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some implementations, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body pain, shortness of breath or difficulty breathing, loss of smell (hypophidrosis, anosmia), loss of taste (hypophidrosis, anosmia), and / or multiple inflammatory syndrome in children or adults (MIS-C, MIS-A). In some embodiments, the level is higher than that of the control composition by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0340] In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the transmission of one or more strains (variants) of a pathogen from one subject to another at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the colonization of one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the colonization of one or more strains (variants) of a pathogen in the nasopharynx of the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits asymptomatic infection of one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein inhibits the replication of one or more strains (variants) of a pathogen in the subject and / or reduces the pathogen load of one or more strains (variants) of a pathogen in the subject at a level higher than that of a control composition. In some embodiments, the level is higher than that of the control composition by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23.

[0341] In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to subjects between about 6 weeks of age and about 5 years of age (e.g., before the 6th birthday) for active immunization to prevent disease caused by one or more strains (variants) of a pathogen. In some embodiments, one or more strains (variants) of such pathogens are selected from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Serum types 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

[0342] In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to subjects aged approximately 6 to approximately 17 years (e.g., before their 18th birthday) for active immunization to prevent disease caused by one or more strains (variants) of a pathogen. In some embodiments, one or more strains (variants) of such pathogens are selected from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Serum types 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

[0343] In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to subjects aged 18 years or older for active immunization to prevent disease caused by one or more strains (variants) of a pathogen. In some embodiments, one or more strains (variants) of such pathogens are selected from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Serum types 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

[0344] antibody composition

[0345] Some embodiments provide an antibody composition comprising antibodies produced in mammals immunized with an immunogenic agent or immunogenic composition of the present disclosure. In some embodiments, the antibody comprises at least one antibody selected from mAbs and anti-idiotype antibodies. In some embodiments, the antibody composition comprises a neutralizing antibody. In some embodiments, the antibody composition comprises isolated gamma globulin fractions. In some embodiments, the antibody composition comprises a polyclonal antibody. In some embodiments, the antibody composition is administered to a subject. In some embodiments, the antibody composition administered to a subject confers passive immunization.

[0346] Application method

[0347] In some embodiments, the immunogenic composition (e.g., a vaccine) or pharmaceutical composition described herein is administered to a subject (e.g., an infant, young child, adolescent, or older adult) at risk of developing a disease due to one or more pathogens. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks and about 6 weeks old. In some embodiments, the human is between about 6 weeks and about 6 years old. In some embodiments, the human is between about 6 years and about 18 years old. In some embodiments, the human is between about 18 years and about 50 years old. In some embodiments, the human is about 50 years old or older. In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein are administered to subjects at elevated risk of developing a disease due to one or more pathogens, such as subjects with immunodeficiency, sickle cell disease or other hemoglobinopathies, congenital or acquired asplenia, splenic dysfunction, chronic renal failure or nephrotic syndrome, diseases associated with treatments involving immunosuppressive drugs or radiation therapy (including malignancies, leukemia, lymphoma, Hodgkin's disease, or solid organ transplantation), congenital or acquired immunodeficiency, HIV infection, cerebrospinal fluid leakage, cochlear implantation, chronic heart disease, chronic lung disease, diabetes, alcoholism, chronic liver disease, smoking, asthma, systemic malignancies, multiple myeloma, or solid organ transplantation. It should be understood that a subject may be considered at risk of developing a disease even if they do not exhibit any symptoms of a disease. For example, a subject is considered at risk of developing a disease if they are known to be or intend to be in a situation with a relatively high risk of infection.

[0348] The immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein can be administered via any effective route of administration, such as oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, transdermal, intradermal, rectal, vaginal, local, ocular, pulmonary, or contact application. In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions can be injected (e.g., via intramuscular, intraperitoneal, intradermal, and / or subcutaneous routes); or delivered via mucosal routes (e.g., to the oral / gastrointestinal tract, respiratory tract, and / or genitourinary tract). In some embodiments, it may be necessary to administer different doses of the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions via different routes; in some embodiments, it may be necessary to administer a dose of a different component via different routes. In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions disclosed herein are administered intramuscularly. In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions disclosed herein are administered subcutaneously.

[0349] In some implementations, an immunogenic composition (e.g., a vaccine) or a pharmaceutical composition is administered intradermally. The standard technique for intradermal injection, the "Mantoux procedure," involves the following steps: cleaning the skin, then stretching it with one hand and inserting the needle at an angle between 10 and 15° with the bevel of a narrow-gauge needle (26-31 gauge) facing upwards. Once the bevel of the needle is inserted, the syringe is lowered and advanced further while applying slight pressure to elevate it under the skin. The liquid is then injected very slowly, creating a blister or bump on the skin surface, followed by slow withdrawal of the needle.

[0350] Devices specifically designed for applying liquid agents to or onto the skin have been described, such as those described in WO 99 / 34850 and EP 1092444, and others such as WO 01 / 13977; US Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,7...

Claims

1. A composition comprising purified capsular polysaccharide having repeating units of Formula I: I Each R is independently selected from H and -C(O)-CH3.

2. The composition of claim 1, wherein the purified capsular polysaccharide comprises about 4 to about 4000 repeating units of formula I.

3. The composition of claim 1 or 2, wherein the purified capsular polysaccharide comprises repeating units between 0% and 100%, wherein R is -C(O)-CH3.

4. The composition according to any one of claims 1-3, wherein the weight-average molecular weight of the purified capsular polysaccharide is about 900 Daltons to about 4,000 kilodaltons.

5. The composition of any one of claims 1-4, wherein the purified capsular polysaccharide is activated with a chemical reagent to generate a reactive group for conjugation with a suitable conjugate (e.g., a linker or carrier protein).

6. The composition of claim 5, wherein activation occurs in β-Gal f At residues C2, C3, C5 or C6.

7. A composition comprising a polysaccharide having one or more monomers of formula II: II.

8. An immunogenic composition comprising (i) a purified capsular polysaccharide and (ii) a polypeptide, wherein the purified capsular polysaccharide comprises repeating units of formula I: I Each R is independently selected from H and -C(O)-CH3.

9. The immunogenic composition of claim 8, wherein the purified capsular polysaccharide comprises about 4 to about 4000 repeating units of formula I.

10. The immunogenic composition of claim 8 or 9, wherein the purified capsular polysaccharide comprises repeating units between 0% and 100%, wherein R is -C(O)-CH3.

11. The immunogenic composition of claim 8, 9 or 10, wherein the purified capsular polysaccharide comprises about 60% to about 70% repeating units, wherein R is -C(O)-CH3.

12. The immunogenic composition according to any one of claims 8-11, wherein the polypeptide is associated with the purified capsular polysaccharide.

13. The immunogenic composition of any one of claims 8-12, wherein the polypeptide is a carrier protein, optionally wherein the carrier protein is a toxoid or a glycoprotein derived from a pathogen, optionally wherein the carrier protein is selected from the group consisting of CRM197, tetanus toxoid, exoprotein A, PspA, diphtheria toxoid, and protein D.

14. The immunogenic composition according to any one of claims 8-12, wherein the purified capsular polysaccharide having one or more repeating units of formula I is biotinylated.

15. The immunogenic composition of claim 14, wherein the polypeptide is a fusion protein comprising a biotin-binding moiety and at least one polypeptide antigen, and wherein a biotinylated purified capsular polysaccharide is non-covalently associated with the biotin-binding moiety of the fusion protein.

16. The immunogenic composition according to any one of claims 8-15, wherein the immunogenic composition further comprises Streptococcus pneumoniae (… Streptococcus pneumoniae One or more polysaccharides, each having an independently selected serotype from the following: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18 C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A , 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F and 48.

17. A pharmaceutical composition comprising the immunogenic composition as described in any one of claims 8-16, and a pharmaceutically acceptable carrier.

18. A method for inducing resistance against Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae A method for an immune response to capsular polysaccharides, comprising administering to a subject the immunogenic composition as described in any one of claims 8-16 or the pharmaceutical composition as described in claim 17.

19. An activated capsular polysaccharide comprising one or more units of formula I, I Each R is independently selected from H and -C(O)-CH3.

20. An activated capsular polysaccharide comprising one or more units of formula I, I Each R is independently selected from H and -C(O)-CH3, and the purified capsular polysaccharide is conjugated with a carrier protein or biotin.

21. A purified capsular polysaccharide comprising one or more monomers of formula III: III One of the R a It is CN or -C(NH)NR b And R b These are proteins (e.g., carrier proteins) and biotin, as well as the rest of the R... a Each is H.

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