Immunogenic compositions

Abstract

Technologies for providing immunogenic compositions (e.g., vaccines) and methods of inducing immune responses in subjects in need thereof.

Description

IMMUNOGENIC COMPOSITIONSCross Reference to Related Applications

[0001] The present application claims the benefit of U.S. Prov. Appln. No. 63 / 452,681 filed March 16, 2023, the entire contents of which are incorporated by reference herein.Background

[0002] The vast majority of vaccines manufactured worldwide each year are given to babies, children, and adults. Typically, vaccines contain excipients, some of which may be added for a specific purpose. Certain common excipients present in vaccines include preservatives, adjuvants, stabilizers, etc.

[0003] As an example, adjuvants are generally added to help stimulate a stronger immune response. Aluminum-containing adjuvants are vaccine ingredients that have been used in vaccines since the 1930s. Surprisingly, despite being widely employed, the mechanisms behind the immune stimulating properties of aluminum-based adjuvants are yet to be determined.Summary

[0004] Among other things, the present disclosure provides insights and technologies that are useful for providing immunogenic compositions (e.g., vaccines). In some embodiments, insights and technologies provided herein can be useful to provide immunogenic compositions (e.g., vaccines) with increased immunogenicity to protect against certain diseases or disorders. In particular embodiments, insights and technologies described herein are useful for MAPS -based immunogenic compositions (e.g., vaccines).

[0005] The present Applicant has previously employed MAPS technologies to develop multivalent vaccines, in which at least one or more polysaccharide antigens are non- covalently associated with one or more polypeptide antigens, for example, in some embodiments, multivalent pneumococcal vaccines as described in WO 2020 / 056202 and PCT / US2022 / 043156, the relevant contents of which are hereby incorporated herein by reference in their entirety for purposes described herein.

[0006] Among other things, the present disclosure provides an insight that the immunogenicity of MAPS immunogenic compositions can be improved by varying the types and / or concentrations of certain excipients (including, e.g., but not limited to adjuvants) and / or formulation buffers used in the MAPS immunogenic compositions. For example, in one aspect, while aluminum-based adjuvant has been widely used in vaccines to enhance the ability to provoke an immune response against and improve the overall potency of the vaccines, the present disclosure, among other things, provides an insight that it may be desirable, especially for MAPS immunogenic compositions, to have a reduced content of aluminum-based adjuvant. Without wishing to be bound by any particular theory, a reduced content of aluminum-based adjuvant, in some embodiments, is characterized by the amount of aluminum present in a MAPS immunogenic composition, while in some embodiments is characterized by the ratios of aluminum content to polysaccharide antigen content and / or polypeptide antigen content. In particular embodiments, the present disclosure has demonstrated that a MAPS immunogenic composition comprising an aluminum-based adjuvant, at a concentration of aluminum that is less than 1.25 mg / mL in the composition, can be particularly useful in increasing an immune response (e.g., in some embodiments, a B cell immune response and / or a T cell immune response) against polysaccharide antigen(s) and / or polypeptide antigen(s). In some embodiments, the present disclosure has demonstrated that a MAPS immunogenic composition comprising an aluminum-based adjuvant, wherein a ratio of the mass of aluminum (from the aluminum-based adjuvant) to the total mass of polysaccharide antigen(s) from MAPS is 3.3:1 in the composition, can be particularly useful in increasing an immune response (e.g., in some embodiments, a B cell immune response and / or a T cell immune response) against polysaccharide antigen(s) and / or polypeptide antigen(s). In some embodiments, the present disclosure has demonstrated that a MAPS immunogenic composition comprising an aluminum-based adjuvant, wherein a ratio of the mass of aluminum (from the aluminum-based adjuvant) to the total mass of fusion protein(s) from MAPS is 1.1:1 in the composition, can be particularly useful in increasing an immune response (e.g., in some embodiments, a B cell immune response and / or a T cell immune response) against polysaccharide antigen(s) and / or polypeptide antigen(s). In some embodiments, the present disclosure has demonstrated that a MAPS immunogenic composition comprising an aluminum-based adjuvant, wherein a ratio of the mass of aluminum (from the aluminum-based adjuvant) to the total mass of MAPS immunogenic complexes is 0.8: 1 in the composition, can be particularly useful in increasing an immuneresponse (e.g., in some embodiments, a B cell immune response and / or a T cell immune response) against polysaccharide antigen(s) and / or polypeptide antigen(s).

[0007] Among other things, and without wishing to be bound by any particular theory, the present disclosure provides an insight that the presence of MAPS complexes in free form (i.e., MAPS complexes not associated with aluminum-based adjuvant) may be desirable in inducing an immune response against an antigen, as compared to MAPS complexes associated with aluminum-based adjuvant.

[0008] In some embodiments, the present disclosure, among other things, provides an insight that a reduced content of aluminum-based adjuvant may be useful for increasing the immunogenicity of MAPS immunogenic compositions (e.g., vaccines) when they are administered to certain patient subjects, for example, in certain embodiments to adult subjects. In some embodiments, such an immune response can be a B cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response can be a T cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response can be a B cell immune response and a T cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens).

[0009] In some embodiments, the present disclosure, among other things, recognizes that certain formulation buffers may be more desirable for use in MAPS immunogenic compositions (e.g., vaccines). In some embodiments, a succinate buffer may be desirable for use in such MAPS immunogenic compositions.

[0010] Accordingly, the present disclosure, among other things, provides technologies (e.g., compositions, methods, and / or kits) for inducing an immune response in subjects in need thereof. In particular embodiments, such technologies are useful for MAPS immunogenic compositions.

[0011] One aspect of the present disclosure provides a pharmaceutical composition comprising: an immunogenic composition comprising one or more species of immunogenic complexes, wherein the immunogenic complex of at least one of the species comprises: (a) a biotinylated polysaccharide antigen; and (b) a fusion protein comprising: (i) a biotin-binding moiety; and (ii) at least one polypeptide antigen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein. Insome embodiments, the pharmaceutical composition further comprises an adjuvant that is or comprises an aluminum-based adjuvant, at a reduced amount of aluminum in the pharmaceutical composition.

[0012] Another aspect of the present disclosure provides a method of making a pharmaceutical composition described herein. For example, in some embodiments, such a method comprises a step of combining an immunogenic composition with an adjuvant being or comprising an aluminum-based adjuvant to form a pharmaceutical composition that has a reduced amount of aluminum, wherein the immunogenic composition comprises one or more species of immunogenic complexes, wherein the immunogenic complex of at least one of the species comprises: (a) a biotinylated polysaccharide antigen; and (b) a fusion protein comprising: (i) a biotin-binding moiety; and (ii) at least one antigenic polypeptide; and wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein.

[0013] In some embodiments, the reduced amount of aluminum in pharmaceutical compositions described herein can be expressed as the mass amount of elemental aluminum (from an aluminum-based adjuvant) relative to the solution volume of the respective pharmaceutical compositions. In some embodiments, the reduced amount of aluminum in a pharmaceutical composition described herein can be reflected by a ratio of mass or molar amount of elemental aluminum to the total mass or molar amount of biotinylated polysaccharide antigen(s) present in the pharmaceutical composition. Tn some embodiments, the reduced amount of aluminum in a pharmaceutical composition described herein can be reflected by a ratio of mass or molar amount of elemental aluminum to the total mass or molar amount of fusion protein(s) present in the pharmaceutical composition. In some embodiments, the reduced amount of aluminum in a pharmaceutical composition described herein can be reflected by a ratio of mass or molar amount of elemental aluminum to the total mass or molar amount of immunogenic complexes present in the pharmaceutical composition.

[0014] In some embodiments of various aspects described herein, the concentration of aluminum in the pharmaceutical composition is reduced relative to a reference composition. In some embodiments, the pharmaceutical composition is characterized in that, upon administration to a subject, it induces an immune response to (i) at least one of the polysaccharide antigens and / or (ii) at least one of the polypeptide antigens, at a level that ishigher than a corresponding reference level, wherein the reference level is a level of an immune response induced in a subject following administration of a reference composition. In some embodiments, the immune response is or comprises an antibody and / or B cell response.

[0015] In some embodiments of various aspects described herein, the immune response is at a level that is at least 20% higher than the corresponding reference level as observed in a reference composition. In some embodiments, the reference composition comprises the immunogenic composition and an adjuvant that is or comprises an aluminum- based adjuvant, wherein the concentration of aluminum in the reference composition is 1.25 mg / mL or greater. In some embodiments, the reference composition is formulated for administration to a human. In some embodiments, the pharmaceutical composition is formulated for administration to a human.

[0016] In some embodiments of various aspects described herein, the concentration of aluminum in the pharmaceutical composition is less than 1.25 mg / mL, e.g., about 0.125 mg / mL to about 1.25 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is about 0.25 to less than 1.25, about 0.25 to about 1, about 0.3 to about 0.85, or about 0.35 to about 0.75 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is less than 0.25 mg / mL, e.g., about 0.125 mg / mL to about 0.25 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is about 0.35 mg / mL to about 0.65 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is about 0.4 to about 0.6 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is about 0.5 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is at least 0.35 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is at least 0.25 mg / mL. In some embodiments, the concentration of aluminum in the pharmaceutical composition is about 0.125 mg / mL, about 0.150 mg / mL, about 0.175 mg / mL, about 0.2 mg / mL, about 0.225 mg / mL, about 0.25 mg / mL, about 0.275 mg / mL, about 0.3 mg / mL, or about 0.325 mg / mL.

[0017] In some embodiments of various aspects described herein, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is less than 3.68:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in thepharmaceutical composition is about 3.68:1 to about 1:1, about 2.5:1 to about 1.1:1, about 2.25 to about 1.2:1, about 2: 1 to about 1.3: 1, about 1.75:1 to about 1.25:1, or about 1.6: 1 to about 1.4:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 1.91:1 to about 1.03:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 1.3: 1 to about 1.6: 1 or about 1.4: 1 to about 1.5:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 1.47:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is at least 1.03: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is at least 0.73:1.

[0018] In some embodiments of various aspects described herein, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is less than 1.22:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is about 0.64: 1 to about 0.34: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is about 0.4:1 to about 0.6:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is about 0.49:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is at least 0.34:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is at least 0.24: 1.

[0019] In some embodiments of various aspects described herein, the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is less than 0.92:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is about 0.48: 1 to about 0.26: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is about 0.5:1 to about 0.3:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is about 0.37:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the immunogeniccomplexes in the pharmaceutical composition is at least 0.26:1. In some embodiments, the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is at least 0.18: 1.

[0020] In some embodiments of various aspects described herein, an aluminum-based adjuvant included in pharmaceutical compositions described herein is or comprises aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate (alum), or a combination thereof. In some embodiments, the aluminum-based adjuvant is aluminum phosphate. In some embodiments, the concentration of the aluminum phosphate in the pharmaceutical composition is less than 5.65 mg / mL. In some embodiments, the concentration of the aluminum phosphate in the pharmaceutical composition is about 2.94 mg / mL to about 1.58 mg / mL. In some embodiments, the concentration of aluminum phosphate in the pharmaceutical composition is about 1 mg / mL to about 4 mg / mL or about 2 mg / mL to about 3 mg / mL. In some embodiments, the concentration of aluminum phosphate in the pharmaceutical composition is about 2.26 mg / mL. In some embodiments, the concentration of the aluminum phosphate in the pharmaceutical composition is at least 1.58 mg / mL. In some embodiments, the concentration of the aluminum phosphate in the pharmaceutical composition is at least 1.13 mg / mL.

[0021] In some embodiments of various aspects described herein, a biotinylated polysaccharide antigen included in an immunogenic complex described herein comprises a bacterial polysaccharide antigen, a fungal polysaccharide antigen, a parasitic polysaccharide antigen, a viral polysaccharide antigen, a mammalian polysaccharide antigen (e.g., a tumor polysaccharide antigen). In some embodiments, the biotinylated polysaccharide antigen comprises a polysaccharide antigen associate with a pathogen (e.g., bacteria, fungi, parasite, or virus). In some embodiments, the biotinylated polysaccharide antigen comprises a polysaccharide antigen associated with a tumor. In some embodiments, the polysaccharide antigen is or comprises a polysaccharide selected from the group consisting of: Salmonella polysaccharide, pneumococcal polysaccharides, Haemophili polysaccharides, meningococcal polysaccharides, staphylococcal polysaccharides, Bacillus anthracis polysaccharide, Streptococcus polysaccharide, Pseudomonas polysaccharide, Klebsiella polysaccharide, Cryptococcus polysaccharide, other bacterial capsular or cell wall polysaccharides, viral polysaccharides (e.g., viral glycoproteins), or combinations thereof.

[0022] In some embodiments of various aspects described herein, a biotinylated polysaccharide antigen included in an immunogenic complex described herein comprises one or more polysaccharides from Streptococcus pneumoniae. In some embodiments, the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9E, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, HD, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48. In some embodiments, the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, HA, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38. In some embodiments, the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from 6C, 7C, 15A, 16F, 23 A, 23B, 24F, 31, 35B, and 38.

[0023] In some embodiments of various aspects described herein, at least one of polypeptide antigen(s) included in an immunogenic complex described herein is or comprises an antigen selected from the group consisting of: bacterial polypeptide antigens, fungal polypeptide antigens, parasitic polypeptide antigens, viral polypeptide antigens, and mammalian polypeptide antigens (e.g., tumor antigens). In some embodiments, at least one of the polypeptide antigen(s) is or comprises a polypeptide antigen associated with a pathogen (e.g., bacteria, fungi, parasite, or virus). In some embodiments, at least one of the polypeptide antigen(s) is or comprises a polypeptide antigen associated with a tumor.

[0024] In some embodiments of various aspects described herein, at least one of the polypeptide antigen(s) included in an immunogenic complex described herein is or comprises an antigen selected from the group consisting of: streptococcal antigens (e.g., 5. pneumoniae, group A, group B, and viridans antigens), tuberculosis antigens, tetanus antigens, anthrax antigens, pertussis antigens, staphylococcal antigens (e.g., S. aureus), Haemophilus antigens, Enterobacter, antigens, Acinetobacter antigens, Citrobacter antigens, Serratia antigens, Clostridia antigens, Campylobacter antigens, Vibriocholera antigens, Pseudomonas antigens,meningococcal antigens, Neisseria gonorrhoeae antigens, Chlamydia trachomatis antigens, Klebsiella antigens, Shigella antigens, Salmonella antigens, E. coli antigens, malaria antigens, HIV antigens, HPV antigens, influenza (e.g., seasonal or epidemic) antigens, coronavirus antigens (e.g., SARS-CoV-2 antigens), herpes (e.g., HSV) antigens, tumor antigens, and combinations thereof. In some embodiments, at least one of the polypeptide antigens is or comprises a pneumococcal polypeptide antigen. In some embodiments, at least one of the polypeptide antigens is or comprises a pneumolysin polypeptide antigen, a SP1500 polypeptide antigen, a SP0785 polypeptide antigen, a SP0435 polypeptide antigen, or a combination thereof.

[0025] In some embodiments of various aspects described herein, at least one species of immunogenic complexes included in pharmaceutical compositions described herein comprises a fusion protein comprising: (i) a biotin-binding moiety; (ii) a pneumolysin polypeptide antigen or antigenic fragment thereof; and (iii) an SP0435 polypeptide antigen or antigenic fragment thereof. In some embodiments, the fusion protein is or comprises an amino acid sequence 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 100% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0026] In some embodiments of various aspects described herein, at least one species of immunogenic complexes included in pharmaceutical compositions described herein comprises a fusion protein comprising: (i) a biotin-binding moiety; (ii) an SPL500 polypeptide antigen or antigenic fragment thereof; and (iii) an SP0785 polypeptide antigen or antigenic fragment thereof. In some embodiments, the fusion protein is or comprises an amino acid sequence 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 100% identical to SEQ ID NO: 30 or SEQ ID NO: 31.

[0027] In some embodiments of various aspects described herein, immunogenic composition included in pharmaceutical compositions described herein 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 species of immunogenic complexes. In some embodiments, the immunogenic composition comprises at least 30, at least 31, at least 32, at least 33, or at least 34 species of immunogenic complexes. In some embodiments, the immunogenic composition comprises nomore than 60, no more than 50, no more than 45, no more than 40, or no more than 35 species of immunogenic complexes.

[0028] In some embodiments of various aspects described herein, one or more species of immunogenic complexes included in pharmaceutical compositions described herein comprise: (i) a first plurality of species of immunogenic complexes comprising: biotinylated polysaccharide antigens selected from each of Streptococcus pneumoniae serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F, wherein each biotinylated polysaccharide antigen is non-covalently complexed with a biotin-binding moiety of a first fusion protein: wherein the first fusion protein comprises: (a) a biotin-binding moiety; (b) a first polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 7 or an antigenic fragment thereof; and (c) a second polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 9 or an antigenic fragment thereof; and (ii) a second plurality of species of immunogenic complexes comprising: biotinylated polysaccharide antigens selected from each of Streptococcus pneumoniae serotypes 2, 3, 5, 6C, 7C, 7F, 8, 9N, 10A, HA, 12F, 14, 15A, 16F, 17F, 19F, 20B, 22F, 23A, 23B, 24F, 31, 35B, and 38, wherein each biotinylated polysaccharide antigen is non-covalently complexed with a biotin-binding moiety of a second fusion protein: wherein the second fusion protein comprises: (a) a biotin-binding moiety; (b) a first polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 13 or an antigenic fragment thereof; and (c) a second polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 11 or an antigenic fragment thereof.

[0029] In some embodiments of various aspects described herein, a biotin-binding moiety included in a fusion protein of immunogenic complexes described herein is or comprises a rhizavidin polypeptide. In some embodiments, the rhizavidin polypeptide is: (i) a polypeptide comprising an amino acid sequence 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%, atleast 99.5%, or 100%’ identical to SEQ ID NO: 1 or a biotin-binding fragment thereof; or (ii) a polypeptide comprising an amino acid sequence 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 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3, or a biotin-binding fragment thereof.

[0030] In some embodiments of various aspects described herein, a pharmaceutical composition described herein is or comprises a vaccine.

[0031] In some embodiments of various aspects described herein, a pharmaceutical composition described herein further comprises one or more of: (a) a buffer (e.g., including but not limited to a histidine buffer; a succinate buffer, and / or a phosphate buffer); (b) a salt (e.g., including but not limited to sodium chloride); (c) a surfactant (e.g., including but not limited to polysorbate 80); and (d) a stabilizer (e.g., including but not limited to sucrose). In some embodiments, a pharmaceutical composition described herein comprises a buffer that is or comprises a succinate buffer.

[0032] Another aspect of the present disclosure provides a dose of a pharmaceutical composition described herein. In some embodiments, the amount of aluminum in the dose is less than 0.625 mg, e.g., between about 0.125 mg and about 0.625 mg. In some embodiments, the amount of aluminum in the dose is between about 0.325 mg and about 0.175 mg. In some embodiments, the amount of aluminum in the dose is about 0.25 mg. In some embodiments, the amount of aluminum in the dose is at least 0. 175 mg. In some embodiments, the amount of aluminum in the dose is at least 0. 125 mg. In some embodiments, the volume of the dose is about 0.5 mL. In some embodiments, the dose is a dose for use in a human.

[0033] In some aspects, delivery devices or containers comprising a dose of a pharmaceutical composition described herein are also within the scope of the present disclosure. For example, in one aspect, provided herein is a syringe comprising a dose of a pharmaceutical composition described herein. In another aspect, provided herein is a vial comprising a pharmaceutical composition described herein or one more of dose(s) of a pharmaceutical composition described herein.

[0034] Technologies (e.g., compositions and methods) described herein are useful for administration to subjects. For example, in some embodiments, technologies (e.g., compositions and methods) described herein are useful for immunization of subjects. Inparticular embodiments, subjects amenable to technologies described herein are human subjects. In some embodiments, human subjects are adult human subjects. In some embodiments, human subjects are pediatric subjects. For example, one aspect of the present disclosure provides a method comprising a step of: administering to the subject an immunologically effective amount of a pharmaceutical composition described herein or a dose of a pharmaceutical composition described herein. Another aspect of the present disclosure provides a method of immunizing a subject, the method comprising a step of: administering to the subject an immunologically effective amount of a pharmaceutical composition described herein or a dose of a pharmaceutical composition described herein. In some embodiments, upon administration, the pharmaceutical composition or the dose induces an immune response to (i) at least one of the polysaccharide antigens and / or (ii) at least one of the polypeptide antigens. In some embodiments, the immune response is or comprises an antibody and / or B cell response. In some embodiments, the immune response is at a level that is higher than a corresponding reference level, wherein the reference level is a level of an immune response induced in a subject following administration of a reference composition. In some embodiments, the immune response is at a level that is at least 20% higher than the corresponding reference level. In some embodiments, the reference composition comprises an immunogenic composition and an adjuvant that is or comprises an aluminum-based adjuvant, wherein the concentration of aluminum in the reference composition is 1.25 mg / mL or greater.Brief Description of the Drawings

[0035] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0036] Figure 1 is a schematic representation of an exemplary MAPS technology, in which MAPS immunogenic complexes comprise one or more polypeptide antigens fused to the biotin-binding protein rhizavidin, or a biotin-binding domain or biotin-binding fragment thereof, and a biotinylated antigenic polysaccharide. In this figure, each MAPS complex isformed between one or more fusion proteins and a biotinylated polysaccharide by non- covalent binding of a truncated rhizavidin to biotin.

[0037] Figure 2 is a schematic of an exemplary CPI fusion protein. Such an exemplary CPI fusion protein comprises a biotin-binding protein such as, e.g., a truncated rhizavidin protein (e.g., amino acids 45-179 of a wild-type rhizavidin protein), a first linker (e.g., a GGGGSSS (SEQ ID NO: 38) linker), a SP1500 polypeptide (e.g. , amino acids 27-278 of a full-length S. pneumoniae SP1500 polypeptide), a second linker (e.g., the amino acid sequence AAA), and a SP0785 polypeptide (e.g. , amino acids 33-399 of a full length .S'. pneumoniae SP0785 polypeptide). In some embodiments, a CPI fusion protein may further comprise a detectable or purification tag (e.g., His tag). The amino acid sequence AAA can be from the Not I site on a pET21 / 24 plasmid, or synthesized. For a GGGGSSS (SEQ ID NO: 38) linker, the SSS amino acid sequence can be from the Sac I site on a pET21 / 24 plasmid, with the GGGG (SEQ ID NO: 67) amino acid sequence added to create a flexible linker with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO: 38) linker can be synthesized.

[0038] Figure 3 is a schematic of an exemplary SPP2 fusion protein. Such an exemplary SPP2 fusion protein comprises a biotin-binding protein, such as e.g., a truncated rhizavidin protein (e.g., amino acids 45-179 of a wild-type rhizavidin protein, denoted as Rhavi), a first linker (e.g. , a GGGGSSS (SEQ ID NO: 38) linker), a pneumolysin (Ply) polypeptide (e.g., amino acids 2-470 of a full-length S. pneumoniae Ply polypeptide comprising mutations G294P, D385N, C428G, and W433F, denoted as PdT(G294P)), a second linker (e.g., a GGGGSSS (SEQ ID NO: 38) linker), and a SP0435 polypeptide (e.g., amino acids 62-185 of a full length .S'. pneumoniae SP0435 polypeptide). In some embodiments, a SPP2 fusion protein may further comprise a detection or purification tag (e.g., a His tag). For a GGGGSSS (SEQ ID NO: 38) linker, the SSS amino acid sequence can be from the Sac I site on a pET21 / 24 plasmid, with the GGGG (SEQ ID NO: 67) amino acid sequence added to create a flexible linker with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO: 38) linker can be synthesized.

[0039] Figure 4 is a schematic of a plasmid construct composed of the pET-24a(+) vector containing a sequence encoding an exemplary SPP2 fusion protein.

[0040] Figure 5 are flow-charts depicting an exemplary upstream (upper panel) and downstream (lower panel) manufacturing process for fusion proteins as described herein(e.g., CPI and SPP2). An exemplary SPP2 polypeptide is a fusion protein comprising a truncated rhizavidin [amino acids 45-179 of a full-length rhizavidin protein], a first linker (e.g., a GGGGSSS (SEQ ID NO: 38) linker), a pneumolysin (Ply) polypeptide, a second linker (e.g. , a GGGGSSS (SEQ ID NO: 38) linker), and a SP0435 polypeptide. An exemplary CPI polypeptide is a fusion protein comprising a truncated rhizavidin [amino acids 45-179 of a full-length rhizavidin protein], a first linker (e.g., a GGGGSSS (SEQ ID NO: 38) linker), a SP1500 polypeptide, a second linker (e.g. , the amino acid sequence AAA), and a SP0785 polypeptide. SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFF: tangential flow filtration; DS: drug substance.

[0041] Figure 6 is a table showing exemplary structures of S. pneumoniae antigenic polysaccharides of 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.

[0042] Figure 7 is a flow-chart depicting an exemplary manufacturing process for MAPS immunogenic complexes, comprising biotinylation of antigenic polysaccharides and assembly with fusion protein(s) as described herein (e.g., CPI or SPP2).

[0043] Figure 8 is a flow-chart depicting exemplary manufacturing processes for a MAPS vaccine. For example, such exemplary processes can be used to manufacture a MAPS34 vaccine, comprising MAPS immunogenic complexes that comprise capsular polysaccharides from 34 different .S', pneumoniae serotypes. DMAP: 4- Dimethylaminopyridine; PS: polysaccharide, f In-process tests

[0044] Figure 9 is a schematic depicting various multi- valent vaccine candidates with different combinations of 5. pneumoniae polysaccharides (serotypes) with antigenic fusion proteins CPI and SPP2. The immunization group (A, B, C, S, T) corresponding to each vaccine candidate is indicated. “Prevnar 13 Types” includes serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. “30+ Ten New” includes serotypes 6C, 7C, 15A, 16F, 23A, 23B, 24F, 31, 35B, and 38. “Pn-MAPS24v Next Eleven” includes serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F. Group A > MAPS23 (All on CPI) includes Prevnar 13 Types serotypes and Pn-MAPS-24v Next Eleven serotypes without serotype 2. Group B > MAPS34 (All on CPI) includes Prevnar 13 Types serotypes, Pn-MAPS-24v Next Eleven serotypes, and 30+ Ten New serotypes. Group C > MAPS34 (24-CP1 / 10-SPP2) includes Prevnar 13 Types serotypes and Pn-MAPS-24v Next Eleven serotypes on CPI, and 30+ Ten New serotypes on SPP2. Group S > MAPS34 (Selected 24 / 10) includes Prevnar 13 Typesserotypes, Pn-MAPS-24v Next Eleven, and 30+ Ten New serotypes (serotypes 2, 3, 5, 6C, 7C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 16F, 17F, 19F, 20, 22F, 23A, 23B, 24F, 31, 35B, and 38 on CPI; and serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F on SPP2). Group T > MAPS34 (Selected 24 / 10) includes Prevnar 13 Types serotypes, Pn-MAPS-24v Next Eleven serotypes, and 30+ Ten New serotypes (serotypes 2, 3, 5, 6C, 7C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 16F, 17F, 19F, 20, 22F, 23 A, 23B, 24F, 31, 35B, and 38 on CPI; and serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F on SPP2).

[0045] Figure 10 shows representative IgG levels against 34 capsular polysaccharide serotypes in P2 rabbit sera for each of the immunization groups, A, B, C, S and T. The top panel displays results for the first 24 serotypes (also included in Applicant’s previously described vaccine, see WO 2020 / 056202). The bottom panel displays results for an additional 10 serotypes. Results are expressed as IgG levels in arbitrary units (a.u.) for the top figure and in ng / mL for the bottom figure. Each serotype in the graphs is associated with a set of 5 bars, which depict IgG levels for immunization groups in the following order: A, B, C, S, and T.

[0046] Figure 11 shows relative antibody titers against capsular polysaccharides of the indicated serotypes for MAPS-34 (24-CP1 / 10-SPP2) [immunization group C], MAPS-34 (selected 24 / 10) [immunization group S, 0.0625 mg dose of aluminum], and MAPS-34 (selected 24 / 10) [immunization group T, 0.025 mg dose of aluminum], compared to a baseline of MAPS-34 (all-on-CPl ) [immunization group B]. Geometric mean titer (GMT) ratio and 95% confidence intervals are shown on the graph. For each immunization group, a star indicates that a given capsular polysaccharides is complexed with antigenic fusion protein SPP2. Black stars correspond to immunization group C; gray stars correspond to immunization groups S and T. No star indicates that a capsular polysaccharide is complexed with antigenic fusion protein CPI. Each serotype in the graph is associated with a set of 3 circles, depicting GMT ratio for immunization groups in the following order: C, S, and T.

[0047] Figure 12 shows relative antibody titers against capsular polysaccharides of the indicated serotypes for MAPS-34 (selected 24 / 10) [immunization group T, 0.025 mg dose of aluminum], compared to a baseline of MAPS-34 (selected 24 / 10) [immunization group S, 0.0625 mg dose of aluminum]. Geometric mean titer (GMT) ratio and 95% confidence intervals are shown on the graph.

[0048] Figure 13 shows representative IgG levels against the .S', pneumoniae SP1500- SP0785 portion of CPI fusion protein in P0, Pl and P2 rabbit sera from each of immunization groups A, B, C, S and T. Each dot on the graph represents one rabbit. Results are expressed in pg / ml with 95% confidence intervals on the graph and tabulated as geometric means (pg / ml) below the graph. In the graph, each immunization group is associated with a set of 3 vertical clusters of dots representing P0, Pl, and P2, respectively.

[0049] Figure 14 shows representative IgG levels against the S. pneumoniae PdT(G294P) portion of SPP2 fusion protein in P0, Pl and P2 rabbit sera from each of immunization groups A, B, C, S and T. Each dot on the graph represents one rabbit. Results are expressed in pg / ml with 95% confidence intervals on the graph and tabulated as geometric means (pg / ml) below the graph. In the graph, each immunization group is associated with a set of 3 vertical clusters of dots representing P0, Pl, and P2, respectively.

[0050] Figure 15 shows anti-SP1500-SP0785 IgG titers (pg / mL) in P2 rabbit sera from each of immunization groups C, S and T graphed against anti-PdT(G294P) IgG titers from the same immunization groups.

[0051] Figure 16 shows representative half-maximal inhibitory concentration (IC50) of neutralizing antibodies against the hemolytic activity of native pneumolysin, in Pl and P2 rabbit sera from each of immunization groups C, S and T. Each dot on the graph represents one rabbit. Results are expressed as the IC50 (serum dilution) with 95% confidence intervals on the graph and tabulated as geometric means (IC50) below the graph.

[0052] Figure 17 shows anti-PdT(G294P) IgG titers (pg / mL) in P2 rabbit sera from each of immunization groups C, S and T graphed against half-maximal inhibitory concentration (IC50) of pneumolysin neutralizing antibodies from the same immunization groups.

[0053] Figure 18 is a schematic depicting various multi-valent vaccine candidates with different combinations of .S', pneumoniae polysaccharides (serotypes) with antigenic fusion proteins CPI and SPP2. The immunization cohorts (MAPS-24 (All on CPI), MAPS- 34 (All on CPI), and MAPS-34 (24-CP1 / 10-SPP2)) correspond to each vaccine candidate as indicated. All three vaccine candidates contained 24 polysaccharide serotypes on CPI : 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20B, 22F, 23F, and 33F. For MAPS-34 (All on CPI) and MAPS-34 (24-CP1 / 10-SPP2), 10 additionalpolysaccharide serotypes 6C, 7C, 15A, 16F, 23A, 23B, 24F, 31, 35B, and 38, were on either CPI or SPP2, respectively.

[0054] Figure 19A shows representative IgG levels against 24 capsular polysaccharide serotypes in P2 rabbit sera for each of the aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low) for MAPS-24 (All on CPI). Results are expressed as IgG levels in arbitrary units (a.u.). Each serotype in the graph is associated with a set of 3 bars depicting IgG levels for aluminum groups in the following order: A, B, and C.

[0055] Figure 19B shows relative antibody titers against capsular polysaccharides of the indicated serotypes for MAPS-24 (All on CPI), where aluminum Group C: Low and Group B: Med titers were compared to baseline titers of Group A: High. Geometric mean titer (GMT) ratio and 95% confidence intervals are shown on the graph. Each serotype in the graph is associated with a pair of circles, with the first circle representing GMT ratio for Group B and the second circle representing GMT ratio for Group C.

[0056] Figure 20 shows representative IgG levels against the S. pneumoniae SP1500- SP-0785 portion of CPI fusion protein in Pl and P2 rabbit sera from MAPS-24 (All on CPI) in the three aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low).

[0057] Figure 21A shows representative IgG levels against 34 capsular polysaccharide serotypes in P2 rabbit sera for each of the aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low) for MAPS-34 (All on CPI). Results are expressed as IgG levels in arbitrary units (a.u.). Each serotype in the graph is associated with a set of 3 bars depicting IgG levels for aluminum groups in the following order: A, B, and C.

[0058] Figure 21B shows relative antibody titers against capsular polysaccharides of the indicated serotypes for MAPS-34 (All on CPI), where aluminum Group C: Low and Group B: Med titers were compared to baseline titers of Group A: High. Geometric mean titer (GMT) ratio and 95% confidence intervals are shown on the graph. Each serotype in the graph is associated with a pair of circles, with the first circle representing GMTR for Group B and the second circle representing GMTR for Group C.

[0059] Figure 22 shows representative IgG levels against the 5. pneumoniae SP1500- SP-0785 portion of CPI fusion protein in Pl and P2 rabbit sera from MAPS-34 (All on CPI)in the three aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low)

[0060] Figure 23A shows representative IgG levels against 34 capsular polysaccharide serotypes in P2 rabbit sera for each of the aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low) for MAPS-34 (24-CP1 / 10-SPP2). Results are expressed as IgG levels in arbitrary units (a.u.). Each serotype in the graph is associated with a set of 3 bars depicting IgG levels for aluminum groups in the following order: A, B, and C.

[0061] Figure 23B shows relative antibody titers against capsular polysaccharides of the indicated serotypes for MAPS-34 (24-CP1 / 10-SPP2), where aluminum Group C: Low and Group B: Med titers were compared to baseline titers of Group A: High. Geometric mean titer (GMT) ratio and 95% confidence intervals are shown on the graph. Each serotype in the graph is associated with a pair of circles, with the first circle representing GMTR for Group B and the second circle representing GMTR for Group C.

[0062] Figure 24A shows representative IgG levels against the 5. pneumoniae SP1500-SP-0785 portion of CPI fusion protein in Pl and P2 rabbit sera from MAPS-34 (24- CP1 / 10-SPP2) in the three aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low).

[0063] Figure 24B shows representative IgG levels against the S. pneumoniae PdT(G294P) portion of SPP2 fusion protein in Pl and P2 rabbit sera from MAPS-34 (24- CP1 / 10-SPP2) in the three aluminum formulation groups (Group A: High, Group B: Med, and Group C: Low).Certain Definitions

[0064] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0065] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0066] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastrical, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g. , a plurality of doses separated in time) and / or periodic (e.g. , individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0067] Agent: In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced throughaction of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0068] Amino acid: In its broadest sense, the term “amino acid”, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an 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 the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino- terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from 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.

[0069] Antigen: The term “antigen”, as used herein, 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 by an MHC molecule) or to an antibody. In some embodiments, an antigen induces a humoral response (e.g., including 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 a humoral response and a cellular response. In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biologic polymer (e.g., other than a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a polysaccharide. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigencontaining source). In some embodiments, antigens utilized in accordance with the present disclosure are provided in a crude form. In some embodiments, an antigen is a recombinant antigen. In some embodiments, an antigen is a polypeptide or a polysaccharide that, upon administration to a subject, induces a specific and / or clinically relevant immune response to such polypeptide or polysaccharide. In some embodiments, an antigen is selected to induce a specific and / or clinically relevant immune response to such polypeptide or polysaccharide. As will be appreciated by a skilled artisan, an antigen can 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.

[0070] Associated with: Two entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of affinity interactions, electrostaticinteractions, hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0071] Biotin-binding moiety: The term “biotin-binding moiety”, as used herein, refers to a biotin-binding protein, a biotin-binding fragment thereof, or a biotin-binding domain thereof.

[0072] Binding : It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0073] Carrier protein: As used herein, the term “carrier protein” refers to a protein or peptide that is coupled, or complexed, or otherwise associated with a hapten e.g., a small peptide or lipid) or less immunogenic antigen e.g. , a polysaccharide) and that induces or improves an immune response to such a coupled, or 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 to a hapten or less immunogenic antigen that is coupled, or complexed, or otherwise associated with such a carrier protein. In some embodiments, such an immune response is or comprises a response to both a carrier protein and a hapten or less immunogenic antigen that is coupled, or complexed, or otherwise associated with such a carrier protein. In some embodiments, no significant immune response to a carrier protein itself occurs. In some embodiments, immune response to a carrier protein may be detected; in some embodiments, immune response to such a carrier protein is strong. In some embodiments, a carrier protein is coupled, or complexed, or otherwise associated with one or more other molecules.

[0074] Colonization: As used herein, the term “colonization” generally refers to the ability of a microbe to grow at a target site or surface. For example, the terms “colonization” refers to the ability of a microbe (e.g. , a bacterium) to grow at an anatomical site (e.g. , a mucosal membrane, gastrointestinal tract, injury site, organ, etc. ) of a host.

[0075] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g. , all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g. , as part of a single chemical complex or covalent entity).

[0076] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, a-helix character, -sheet character, coiled-coil character, random coil character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0077] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g. , a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, suchquantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0078] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. 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 comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population i.e., is a therapeutic dosing regimen).

[0079] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment includes a discrete portion of the whole which discrete portion shares one or more functional characteristics found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment of a polymer, e.g., a polypeptide or a polysaccharide, comprises or consists 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 monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units e.g., residues) found in the whole polymer. The whole material or entity may, in some embodiments, be referred to as the “parent” of the whole.

[0080] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are 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% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are 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% similar (e.g. , containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0081] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are 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% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences,for example, can be performed by aligning the two sequences for optimal comparison purposes (<?.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison 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 a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g. , nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0082] Improve, increase, inhibit or reduce: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (<?.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0083] Immunologically effective amount or immunologically effective dose: As used herein, “immunologically effective amount” or “immunologically effective dose” refersto an amount of an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, which when administered to a subject, either in a single dose or as part of a series of doses, that is sufficient to enhance a subject’s own immune response against a subsequent exposure to a pathogen. In some embodiments, the pathogen is S. pneumoniae. In some embodiments, the immune response is against one or more different serotypes of S. pneumoniae. In some embodiments, the immune response is against two or more different serotypes of .S'. pneumoniae. In some embodiments, the immune response is against nine or more different serotypes of .S', pneumoniae. In some embodiments, the immune response is against thirteen or more different serotypes of S. pneumoniae. In some embodiments, the immune response is against fifteen or more different serotypes of .S', pneumoniae. In some embodiments, the immune response is against twenty-three or more different serotypes of .S', pneumoniae. In some embodiments, the immune response is against twenty-four or more different serotypes of .S', pneumoniae. An immunologically effective amount may vary based on the subject to be treated, the species of the subject, the degree of immune response desired to induce, etc. In some embodiments, an immunologically effective amount is sufficient for treatment or protection of a subject having or at risk of having disease. In some embodiments, an immunologically effective amount refers to a non-toxic but sufficient amount that can be an amount 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, an immunologically effective amount is sufficient to induce an immunoprotective response upon administration to a subject.

[0084] Immunoprotective response or protective response: As used herein, “immunoprotective response” or “protective response” refers to an immune response that mediates antigen or immunogen-induced immunological memory. In some embodiments, an immunoprotective response is induced by the administration of a substance, e.g. , an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition to a subject. In some embodiments, immunoprotection involves one or more of active immune surveillance, a more rapid and effective response upon immune activation as compared to a response observed in a naive subject, efficient clearance of the activating agent or pathogen, followed by rapid resolution of inflammation. In some embodiments, an immunoprotective response is an adaptive immune response. In some embodiments, animmunoprotective response is sufficient to protect an immunized subject from productive infection by a particular pathogen or pathogens to which a vaccine is directed (e.g. , S. pneumoniae infection).

[0085] Immunization: As used herein, “immunization”, or grammatical equivalents thereof, refers to a process of inducing an immune response to an infectious organism or agent in a subject (“active immunization”), or alternatively, providing immune system components against an infectious organism or agent to a subject (“passive immunization”). In some embodiments, immunization involves the administration of one or more antigens, immunogens, immunogenic complexes, vaccines, immune molecules such as antibodies, immune sera, immune cells such as T cells or B cells, or pharmaceutical compositions to a subject. In some embodiments, immunization is performed by administering an immunologically effective amount of a substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, immune molecule such as an antibody, immune serum, immune cell such as a T cell or B cell, or pharmaceutical composition to a subject. In some embodiments, immunization results in an immunoprotective response in the subject. In some embodiments, active immunization is performed by administering to a subject an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, vaccine, or pharmaceutical composition. In some embodiments, passive immunization is performed by administering to a subject an immune system component, e.g., an immune molecule such as an antibody, immune serum, or immune cell such as a T cell or B cell.

[0086] Isolated: As used herein, the term “isolated”, or grammatical equivalents thereof, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities 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 they were initially associated. In some embodiments, isolated agents are 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, asubstance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g. , buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polysaccharide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide or polysaccharide. Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide or polysaccharide to the extent that it has been separated from other components a) with which it is associated in nature; and / or b) with which it was associated when initially produced.

[0087] Linker: As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, a polypeptide linker 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 length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides e.g. , fusion polypeptides) are known in the art (Holliger et al, 1993; Poljak, 1994).

[0088] Mutation: As used herein, the term “mutation” is used to refer to a difference in the sequence of a nucleic acid or polypeptide as 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” is understood by those of skill in the art to include a difference as compared to a reference, and does not necessarily refer to or imply a change having occurred within any particular sequence of interest.

[0089] Non-inferior: As used herein, the term “non-inferior” in the context of evaluating a test pharmaceutical composition refers to a test pharmaceutical composition that is (e.g. , in terms of immunogenicity and / or functional antibody titer generated by the test composition) at least as effective as a reference composition. In some embodiments, noninferiority is demonstrated when the lower bound of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of a test pharmaceutical composition over a reference composition is at least greater than or equal to 0.4 or above, including, e.g., 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, an immunogenic composition (e.g., vaccine) described herein is non-inferior to a reference vaccine e.g., PCV13, PCV20, or PPSV23) when the lower bound of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) over the reference vaccine is at least greater than or equal to 0.4 or above, including, e.g., 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, an immunogenic composition (e.g., vaccine) described herein is non-inferior to a reference vaccine (e.g. , PCV 13, PCV20, or PPS V23) when the lower bound of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) over the reference vaccine is at least greater than or equal to 0.95 or above, including, e.g., 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, an immunogenic composition (e.g., vaccine) described herein is non-inferior to a reference vaccine (e.g., PCV13, PCV20, or PPSV23) when the seroconversion rates, or percentages of vaccine recipients with immune responses, are above a pre-defined threshold, e.g., the lower bound of the 95% confidence interval for the difference between the percentage of subjects who seroconvert, following immunization with an immunogenic composition (e.g., vaccine) described herein or immunization with the reference vaccine (e.g., PCV13, PCV20, or PPSV23), is greater than - 0.10.

[0090] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable excipients and / or carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0091] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0092] Polysaccharide: The term “polysaccharide” as used herein refers to a polymeric carbohydrate molecule composed of long chains of monosaccharide units bound together by glycosidic, phosphodiester, or other linkages and on hydrolysis give the constituent monosaccharides or oligosaccharides. Polysaccharides range in structure 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).

[0093] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids, e.g., linked to each other by peptide bonds. Those of ordinary skill in the art will appreciate that the term “polypeptide” isintended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (z.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide’" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0094] Prevention: The term “prevent” or “prevention”, as used herein in connection with a disease, disorder, and / or medical condition, refers to reducing the risk of developing the disease, disorder and / or condition, and / or a delay of onset, and / or reduction in frequency and / or severity of one or more characteristics or symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a pre-defined period of time.

[0095] Protein: As used herein, the term “protein” encompasses a polypeptide. Proteins may include moieties other than amino acids e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.Polypeptides may contain 1- amino acids, d- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g. , 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 a polypeptide having 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, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0096] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g. , in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g. , of a human, a mouse, etc.).

[0097] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, subject, population, sample, sequence or value of interest is compared with a reference or control agent, animal, subject, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneouslywith the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0098] Response: As used herein, a “response” to treatment may refer to any beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment. Such alteration may include stabilization of the condition e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and / or improvement in the prospects for cure of the condition, etc. It may refer to a subject’s response or to a tumor’s response. Subject or tumor response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of biomarkers in a sample obtained from a subject, cytology, and / or histology. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of subjects and / or tumors, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.

[0099] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular subject will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments, a reference sample or group of reference samples are from subjects comparable to a particular subject. In some embodiments, relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0100] Serotype: As used herein, the term “serotype”, also referred to as a serovar, refers to a distinct variation within a species of bacteria or virus or among immune cells ofdifferent subjects. These microorganisms, viruses, or cells are classified together based on their cell surface antigens, allowing the epidemiologic classification of organisms to the subspecies level. A group of serovars with common antigens may be referred to as a serogroup or sometimes serocomplex.

[0101] Species: As used herein, the term “species” refers to a distinct immunogenic complex comprising (i) a biotinylated polysaccharide antigen and (ii) a fusion protein comprising a biotin-binding moiety and one or more Streptococcus pneumoniae polypeptide antigens. In some embodiments, a distinct species can differ in one or more of (i) a biotinylated polysaccharide antigen and (ii) a fusion protein comprising a biotin-binding moiety and one or more Streptococcus pneumoniae polypeptide antigens.

[0102] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g. , a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an subject to whom diagnosis and / or therapy is and / or has been administered.

[0103] Superior: As used herein, the term “superior” in the context of evaluating a test pharmaceutical composition refers to a test pharmaceutical composition that performs e.g., in terms of immunogenicity and / or functional antibody titer generated by the test composition) better than a reference composition. In some embodiments, superiority is demonstrated when the upper bound of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of a test pharmaceutical composition over a reference composition is at least 1.3 or above, including, e.g., at least 1.4, at least 1.5, at least 2, at least 2.5, at least 3, at least 4, or higher. For example, in some embodiments, an immunogenic composition (e.g., vaccine) described herein is superior to a reference vaccine (e.g., PCV13, PCV20, or PPSV23) when the upper bound of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) over the reference vaccine is at least 1.3 or above, including, e.g., at least 1.4, at least 1.5, at least 2, at least 2.5, at least3, at least 4, or higher. In some embodiments, an immunogenic composition (e.g., vaccine) described herein is superior to a reference vaccine (e.g. , PCV 13, PCV20, or PPSV23) when the two-sided 95% confidence interval (CI) for the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) over the reference vaccine excludes zero.

[0104] Susceptible to: A subject who is “susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition is a subject who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and / or condition is a population-based risk (e.g. , family members of subjects suffering from the disease, disorder, or condition).

[0105] Symptoms are reduced: As used herein, “symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g. , intensity, severity, etc.) and / or frequency, e.g., to a statistically and / or clinically significant or relevant level. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

[0106] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0107] Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent. For the purposes of the present disclosure, vaccination can be administered before, during, and / or after exposure to a disease-causing agent, and in certain embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition. In some embodiments, vaccination initiates immunization.

[0108] Variant: As used herein in the context of molecules, e.g. , nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the referencepolypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, 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 the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g. , fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises 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 commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises 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 commonly fewer than about 5, about 4, about 3, or about 2 modifications (e.g., substitutions, additions or deletions) at the N-terminus portion, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises 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 commonly fewer than about 5, about 4, about 3, or about 2 modifications (e.g. , substitutions, additions or deletions) at the C-terminus portion, as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.Detailed Description of Certain Embodiments

[0109] The present disclosure relates, generally, to compositions, systems, and methods that are useful for providing immunogenic compositions (e.g., vaccines). In someembodiments, insights and technologies provided herein can be useful to provide immunogenic compositions (e.g., vaccines) with increased immunogenicity to protect against certain diseases or disorders. In particular embodiments, insights and technologies described herein are useful for MAPS -based immunogenic compositions (e.g., vaccines).

[0110] The present Applicant has previously employed MAPS technologies to develop multivalent vaccines, in which at least one or more polysaccharide antigens are non- covalently associated with one or more polypeptide antigens, for example, in some embodiments, multivalent pneumococcal vaccines as described in WO 2020 / 056202 and PCT / US2022 / 043156, the relevant contents of which are hereby incorporated herein by reference in their entirety for purposes described herein.

[0111] Among other things, the present disclosure provides an insight that the immunogenicity of MAPS immunogenic compositions can be improved by varying the types and / or concentrations of certain excipients (including, e.g., but not limited to adjuvants) and / or formulation buffers used in the MAPS immunogenic compositions. For example, in one aspect, while aluminum-based adjuvant has been widely used in vaccines to enhance the ability to provoke an immune response against and improve the overall potency of the vaccines, the present disclosure, among other things, provides an insight that it may be desirable, especially for MAPS immunogenic compositions, to have a reduced content of aluminum-based adjuvant. Without wishing to be bound by any particular theory, a reduced content of aluminum-based adjuvant, in some embodiments, is characterized by the amount of aluminum present in a MAPS immunogenic composition, while in some embodiments is characterized by the ratios of aluminum content to polysaccharide antigen content and / or polypeptide antigen content. In particular embodiments, the present disclosure has demonstrated that a MAPS immunogenic composition comprising an aluminum-based adjuvant, at a concentration of aluminum that is less than 1.25 mg / mL in the composition, can be particularly useful in increasing an immune response (e.g., in some embodiments, a B cell immune response and / or a T cell immune response) against polysaccharide antigen(s) and / or polypeptide antigen(s).

[0112] Among other things, and without wishing to be bound by any particular theory, the present disclosure provides an insight that the presence of MAPS complexes in free form (i.e., MAPS complexes not associated with aluminum-based adjuvant) may bedesirable in inducing an immune response against an antigen, as compared to MAPS complexes associated with aluminum-based adjuvant.

[0113] In some embodiments, the present disclosure, among other things, provides an insight that a reduced content of aluminum-based adjuvant may be useful for increasing the immunogenicity of MAPS immunogenic compositions (e.g., vaccines) when they are administered to certain patient subjects, for example, in certain embodiments to adult subjects. In some embodiments, such an immune response can be a B cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response can be a T cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response can be a B cell immune response and a T cell immune response (e.g., against one or more polysaccharide antigens and / or polypeptide antigens).

[0114] In some embodiments, the present disclosure, among other things, recognizes that certain formulation buffers may be more desirable for use in MAPS immunogenic compositions (e.g., vaccines). In some embodiments, a succinate buffer may be desirable for use in such MAPS immunogenic compositions.

[0115] Accordingly, the present disclosure, among other things, provides technologies (e.g., compositions, methods, and / or kits) for inducing an immune response in subjects in need thereof. In particular embodiments, such technologies are useful for MAPS immunogenic compositions.Immunogenic Complexes

[0116] The present disclosure encompasses immunogenic complexes that include one or more polypeptides and one or more polymers.

[0117] In some embodiments, immunogenic complexes are, or are based on, MAPS technologies. Aspects of the MAPS technologies have been previously described in W02012 / 155007 and W02020 / 056202, the contents of which are herein incorporated by reference in their entirety, and are shown schematically in Figure 1. See also Zhang et al. , 2013.

[0118] In some embodiments, an immunogenic complex includes one or more polypeptides (e.g., but not limited to, antigenic polypeptides) described herein non-covalentlycomplexed with one or more polymers (e.g., but not limited to, polysaccharides) described herein. In some embodiments, one or more polypeptides (e.g. , but not limited to, antigenic polypeptides) are complexed via affinity interaction with one or more polymers (e.g., but not limited to, polysaccharides). In some embodiments, immunogenic complexes of the disclosure include one or more polypeptides (e.g. , but not limited to, antigenic polypeptides) non-covalently complexed with one or more polymers (e.g., but not limited to antigenic polysaccharides) using one or more affinity molecule pairs each comprising a first affinity molecule and a second affinity molecule complementary to the first affinity molecule (“complementary affinity molecule”). Upon association of the first affinity molecule and the complementary affinity molecule, the one or more polypeptides (e.g., antigenic polypeptides) are non-covalently complexed to the one or more polymers (e.g., antigenic polysaccharides).

[0119] In some embodiments, an immunogenic complex includes (i) one or more polymers (e.g., antigenic polysaccharides) associated with (e.g., in some embodiments, by chemical conjugation) a first affinity molecule described herein, and (ii) a fusion protein that comprises a complementary affinity molecule described herein and one or more polypeptides (e.g., antigenic polypeptides). In some embodiments, an immunogenic complex includes (i) one or more polymers (e.g., antigenic polysaccharides) associated with (e.g., in some embodiments, by chemical conjugation) a plurality of a first affinity molecule described herein, and (ii) a fusion protein that comprises a complementary affinity molecule described herein and one or more polypeptides (e.g., antigenic polypeptides).

[0120] In some embodiments, one or more polypeptides (e.g., antigenic polypeptides) are complexed via affinity interaction with one polymer (e.g. , an antigenic polysaccharide). In some embodiments, immunogenic complexes of the disclosure include one or more polypeptides (e.g., antigenic polypeptides) non-covalently complexed with one polymer (e.g., an antigenic polysaccharide) using one affinity molecule / complementary affinity molecule pair. In some embodiments, immunogenic complexes of the disclosure include one or more polypeptides (e.g., antigenic polypeptides) non-covalently complexed with one polymer (e.g., an antigenic polysaccharide) using one or more affinity molecule / complementary affinity molecule pairs. In some embodiments, each of the affinity molecule / complementary affinity molecule pairs is the same, e.g., biotin / biotin-binding moiety pairs. In some embodiments, an immunogenic complex includes (i) one polymer (e.g., an antigenic polysaccharide) associated with (e.g., in some embodiments, by chemical conjugation) a first affinity molecule describedherein, and (ii) a fusion protein that comprises a complementary affinity molecule described herein and one or more polypeptides (e.g., antigenic polypeptides). In some embodiments, an immunogenic complex includes (i) one polymer (e.g., an antigenic polysaccharide) associated with (e.g., in some embodiments, by chemical conjugation) a plurality of first affinity molecule described herein, and (ii) a fusion protein that comprises a complementary affinity molecule described herein and one or more polypeptides (e.g., antigenic polypeptides). Upon association of the first affinity molecule and the complementary affinity molecule, the one or more polypeptides (e.g., antigenic polypeptides) are non-covalently complexed to the one polymer (e.g., an antigenic polysaccharide).

[0121] In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of biotin / biotin-binding moiety, antibody / antigen, enzyme / substrate, receptor / ligand, metal / metal-binding protein, carbohydrate / carbohydrate binding protein, lipid / lipid-binding protein, and His tag / His tag-binding molecule.

[0122] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is a moiety, e.g., a biotin-binding protein, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is rhizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrAvidin, CaptA vidin™, or a biotin-binding domain or biotin-binding fragment thereof, or a combination thereof. In some embodiments, the biotinbinding moiety is a dimer, e.g., a non-covalent dimer. In some embodiments, the biotinbinding moiety is rhizavidin, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 1, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide that comprises an amino acid sequence that is at least 80% (including, e.g., at least 90%, at least 95%, at least 98%, at least 99%, and 100%) identical to SEQ ID NO: 1, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide that comprises an amino acid sequence that is at least 80% (including, e.g., at least 90%, at least 95%, at least 98%, at least 99%, 100%) identical to SEQ ID NO: 2 or SEQ ID NO: 3, or a biotin-binding domain or biotin-binding fragment thereof.

[0123] In some embodiments, the one or more antigenic polysaccharides are, or are derived from Gram-negative bacteria and / or Gram-positive bacteria. In some embodiments, one or more bacterial antigenic polysaccharides are, or are derived from 5. pneumoniae. In some embodiments, one or more antigenic polysaccharides are, or are derived from one or more pathogens. In some embodiments, one or more antigenic polysaccharides are, or are derived from 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, or 35 serotypes or strains of a pathogen. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 25 serotypes or strains of a pathogen, e.g., 36, 37, 38, 39, 40, 45, 50, 55, or 60 serotypes or strains. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 35 serotypes or strains of a pathogen, e.g., 36, 37, 38, 39, 40, 45, 50, 55, or 60 serotypes or strains. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 70, 80, 90, or 100 serotypes or strains of a pathogen.

[0124] In some embodiments, the one or more antigenic polysaccharides comprise one or more affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the one or more affinity molecules comprise biotin or biotin derivatives.

[0125] In some embodiments, the antigenic polysaccharides comprise a plurality of affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the affinity molecules comprise biotin or biotin derivatives.

[0126] In some embodiments, one or more antigenic polypeptides are covalently linked (e.g. , fused) to a complementary affinity molecule described herein. In some embodiments, a fusion protein comprises one or more antigenic polypeptides 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 rhizavidin or a biotin-binding portion thereof.

[0127] In some embodiments, antigenic polysaccharides and / or antigenic polypeptides that may be included in immunogenic complexes are recombinantly or synthetically produced. In some embodiments, antigenic polysaccharides and / or antigenic polypeptides that may be included in immunogenic complexes are isolated and / or derived from natural sources. In some embodiments, antigenic polysaccharides and / or antigenicpolypeptides that may be included in immunogenic complexes are isolated from bacterial cells. Exemplary polysaccharides and / or polypeptides are described below.

[0128] In some embodiments, an immunogenic complex of the present disclosure is an immunogenic complex as described in WO 2018 / 237221, WO 2020 / 056202, PCT / US2022 / 043156, or PCT / US2022 / 042964. The contents of each of the aforementioned references are incorporated by reference in their entirety for their purposes described herein.Antigenic Polypeptides

[0129] In some embodiments, an immunogenic complex described herein comprises one or more polypeptide antigens. In some embodiments, a polypeptide antigen is or comprises a bacterial polypeptide antigen, a fungal polypeptide antigen, a parasitic polypeptide antigen, a viral polypeptide antigen, and / or a mammalian polypeptide antigen. An exemplary mammalian polypeptide antigen is or comprises a tumor antigen. In some embodiments, a polypeptide antigen is or comprises a polypeptide antigen associated with a pathogen (e.g., bacteria, fungi, parasite, or virus). In some embodiments, a polypeptide antigen is or comprises a polypeptide antigen associated with a tumor.

[0130] In some embodiments, one or more polypeptide antigens included in an immunogenic complex described herein comprise two or more polypeptide antigens from the same source, e.g., in some embodiments, associated with the same pathogen (including, e.g., a bacterium, a fungus, a parasite, a virus, etc.) or associated with the same cancer or tumor. In some embodiments, one or more polypeptide antigens included in a composition described herein comprise polypeptide antigens associated with different pathogens (including, e.g., bacteria, fungi, parasites, viruses, etc.) or associated with a different cancer or tumor. For example, one or more polypeptide antigens included in a composition described herein comprises (i) a polypeptide antigen associated with a first bacterium, fungus, parasite, virus, or cancer or tumor, and (ii) a polypeptide antigen associated with a second bacterium, fungus, parasite, virus, or cancer or tumor.

[0131] In some embodiments, a polypeptide antigen is or comprises a viral polypeptide antigen. In some embodiments, a viral polypeptide antigen is or comprises a coronavirus antigen, an HIV antigen, an HSV (e.g., HSV-1 or HSV-2) antigen, an HPV antigen, an influenza (e.g. , seasonal or epidemic) antigen, or combinations thereof. In someembodiments, a viral polypeptide antigen is a polypeptide antigen of, or derived from, a coronavirus. In some embodiments, a viral polypeptide antigen is a polypeptide antigen of, or derived from, SARS-CoV-2. In some embodiments, a coronavirus polypeptide antigen is a coronavirus Spike (S) protein or antigenic fragment thereof, an Envelope (E) protein or antigenic fragment thereof, a Membrane (M) protein or antigenic fragment thereof, and / or a Nucleocapsid (N) protein or antigenic fragment thereof.

[0132] In some embodiments, a polypeptide antigen is or comprises a fungal polypeptide antigen. In some embodiments, a fungal polypeptide antigen is or comprises an apergillosis antigen, a blastomycosis antigen, a candidiasis antigen, a chromoblastomycosis antigen, a coccidioidomycosis antigen, a emergomycosis antigen, a fungal eye infection antigen, a fungal nail infection antigen, a fusariosis antigen, a histoplasmosis antigen, a mucormycosis antigen, a mycetoma antigen, a paracoccidioidomycosis antigen, a Pneumocystis pneumonia antigen, a ringworm antigen, a scedosporiosis antigen, a sporotrichosis antigen, a talaromycosis antigen, or combinations thereof. In some embodiments, a polypeptide antigen is, or is derived from, an Aspergilus antigen, a Blastomyces antigen, a Candida (e.g., C. auris) antigen, a Coccidioides antigen, a Cryptococcus antigen (e.g., C. neoformans and C. gattii), an Epidermophyton antigen, a Fusarium antigen, a Histoplasma antigen, a Lomentospora antigen, a Madurella antigen, Mucormycetes antigen, a Microsporuni antigen, a Paracoccidioidomycosis antigen, a Pneumocystis (e.g. , P. jirovecii) antigen, a Scedosporium antigen, a Sporothrix antigen, a Talaromyces antigen, a Trichophyton antigen, or combinations thereof.

[0133] In some embodiments, a polypeptide antigen is or comprises a parasitic protozoan polypeptide antigen. In some embodiments, a parasitic protozoan polypeptide antigen is or comprises an amoebiasis antigen, a baesiosis antigen, a blastocystis antigen, a Chagas disease antigen, cryptosporidiosis, a giardiasis antigen, a lambliasis antigen, a malaria antigen, a toxoplasmosis antigen, trichomoniasis antigen, or combinations thereof. In some embodiments, a polypeptide antigen is, or is derived from, a Babesia (e.g. , B. microti) antigen, a Balantidium antigen, a Cryptosporidium antigen, a Entamoeba antigen, a Giardia (e.g. , G. lamblia) antigen, a Leishmania antigen, a Plasmodium (e.g. , P. falciparum) antigen, a Trichomoniasis (e.g., T. vaginalis) antigen, a Trypanosoma (e.g., T. cruzi and T. brucei) antigen, or combinations thereof.

[0134] In some embodiments, a polypeptide antigen is or comprises a tumor antigen. In some embodiments, a tumor antigen is, or is derived from, carcinoembryonic antigen(CEA), cancer / testis antigens (e.g., New York esophageal squamous cell carcinoma 1 (NY- ESO1), mucin-1 (MUC1), and Sialyl Tn (STn)), gangliosides (e.g., GM3 and GD2), p53, HER2 / neu, EGFR (e.g., EGFRvIII), melanocyte / melanoma differentiation antigens (e.g., tyrosinase, MARTI, gplOO, melanoma antigen (MAGE) family proteins, prostate-specific antigen (PSA), leukemia- associated antigens (LAAs) (e.g., BCR-ABL, Wilms’ tumor protein and proteinase 3, idiotype (Id) antibodies, or combinations thereof. See e.g., Mitchell, Curr. Opinion. Investig. Drugs 150 (2002), Dao & Scheinberg, Best Pract. Res. Clin. Haematol. 391 (2008). In some embodiments, a tumor antigen is or comprises a tumor antigen from a solid tumor. In some embodiments, a tumor antigen is or comprises a tumor antigen from a hematologic tumor. In some embodiments, a tumor antigen is or comprises a tumor antigen associated with a cancer. Examples of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, ovarian and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0135] In some embodiments, a polypeptide antigen is or comprises a bacterial polypeptide antigen. In some embodiments, a bacterial polypeptide antigen is a polypeptide antigen of, or derived from, a Gram-negative or a Gram-positive bacterium. In some embodiments, a bacterial polypeptide antigen is or comprises a pneumococcal (e.g., group A, group B, and viridans) antigen, a tuberculosis antigen, an anthrax antigen, a pertussis antigen, a staphylococcal (e.g., S. aureus) antigen, a meningococcal antigen, a haemophilus antigen, a Shigella antigen, a Salmonella antigen, a Pseudomonas antigen, a Klebsiella antigen, an E. coli antigen, or combinations thereof. In some embodiments, a bacterial polypeptide antigen is a polypeptide antigen of, or derived from, S. pneumoniae.

[0136] In some embodiments, a polypeptide antigen is a polypeptide antigen of, or derived from S. pneumoniae. In some embodiments, the one or more polypeptide antigen is a polypeptide antigen of, or derived from, a pathogen other than .S’, pneumoniae. In some embodiments, the one or more polypeptide antigens comprise (i) a polypeptide antigen of, or derived from, S. pneumoniae, and (ii) a polypeptide antigen of, or derived from, a pathogenother than S. pneumoniae. In some embodiments, an immunogenic complex includes one or more of the following 5. pneumoniae antigenic polypeptides, or portions thereof.

[0137] In some embodiments, an immunogenic complex described herein includes one or more of the following antigenic polypeptides, or portions thereof.Exemplary Pneumococcal Polypeptide Antigens: Pneumolysin Polypeptides

[0138] In some embodiments, an antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, a pneumococcal polypeptide antigen is or comprises pneumolysin polypeptide antigen. Pneumolysin (Ply) is a .S', pneumoniae protein toxin. Ply is a cholesterol-dependent toxin of the thiol- activated cytolysin family. In some embodiments, a Ply polypeptide antigen is or comprises a full- length Ply polypeptide. For example, in some embodiments, a full-length Ply polypeptide has 470 amino acids (53 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, a Ply polypeptide antigen includes a portion of a Ply polypeptide (e.g., a portion of a Ply polypeptide of SEQ ID NO: 4, which portion includes 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 contiguous amino acids of SEQ ID NO: 4). In some embodiments, a portion of a Ply polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a Ply polypeptide antigen contains one or more amino acid alterations (e.g., deletion, substitution, and / or insertion) from a naturally-occurring wild-type Ply polypeptide sequence. For example, a Ply polypeptide antigen may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to 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, a Ply polypeptide antigen may contain a portion (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 a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 4. In some embodiments, a nucleotide sequence encoding a Ply polypeptide is provided herein as SEQ ID NO: 14.

[0139] Pneumolysins are exotoxins produced by bacteria that can cause hemolytic activity and complement activation. While highly immunogenic, their use in vaccines can be limited because they cause lysis of red blood cells. Accordingly, in some embodiments, a Ply polypeptide antigen is or comprises a Ply variant (also designated herein as a mutant Ply or “mPly”) that is substantially non-hemolytic. As used herein, the phrase “substantially nonhemolytic” means the ability of lysing red blood cells being reduced by 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%, as compared to that of an equivalent concentration of a reference Ply (e.g., a wildtype Ply). In some embodiments, hemolytic activity of 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%, 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% lower than an equivalent concentration of a reference Ply (e.g., a wild-type Ply). In some embodiments, the substantially non-hemolytic Ply has no detectable hemolytic activity. The term “wild-type Ply” is accorded the usual definition associated with such phrase, e.g., in some embodiments, a naturally occurring Ply (e.g., a Ply that is naturally secreted by a capable bacterial source). In some embodiments, a wild-type Ply protein is represented by the amino acid sequence as set forth in SEQ ID NO: 4.

[0140] In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g. , an amino acid sequence as set forth in SEQ ID NO: 4) or an antigenic fragment thereof, with one or more amino acid substitutions. In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 4) or an antigenic fragment thereof, with one or more of the following amino acid substitutions: residue D385 substituted with N; residue C428 substituted with G, and residue W433 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, a mutant Ply (e.g., non-hemolytic Ply) carrying the amino acid substitutions D385N, C428G, and W433F is referred to as PdT. In some embodiments, a PdT is or comprises the amino acid sequence as set forth in SEQ ID NO: 5.

[0141] In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g. , an amino acid sequence as set forth in SEQ ID NO:4) or an antigenic fragment thereof, with G294 substituted with P (See, e.g., 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, a mutant Ply (e.g., non-hemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 4) or an antigenic fragment thereof, with all of the following amino acid substitutions: residue D385 substituted with N; residue C428 substituted with G; residue W433 substituted with F; and G294 substituted with P. In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) carrying the amino acid substitutions G294P, D385N, C428G, and W433F is referred to as PdT(G294P). In some embodiments, a PdT(G294P) is or comprises the amino acid sequence as set forth in SEQ ID NO: 6.

[0142] In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) is a portion of a PdT(G294P) polypeptide (e.g., a portion of the PdT(G294P) polypeptide of SEQ ID NO: 6, which portion includes 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 contiguous amino acids of SEQ ID NO: 6. In some embodiments, such a portion of PdT(G294P) polypeptide include the four amino acid substitutions described herein. In some embodiments, a portion of a PdT(G294P) polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a mutant Ply (e.g. , non-hemolytic Ply) contains one or more amino acid alterations (e.g. , deletion, substitution, and / or insertion) from the PdT(G294P) polypeptide sequence of SEQ ID NO: 6. For example, a mutant Ply (e.g., non-hemolytic Ply) may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to 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 of the sequence shown in SEQ ID NO: 6). Alternatively, a mutant Ply (e.g., non-hemolytic Ply) may contain a portion (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 a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 6. In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) may comprises no more than 25 (including, 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 than5, no more than 4, no more than 3, no more than 2) amino acid modifications (e.g., deletion, substitution, and / or insertion) within the sequence of SEQ ID NO: 6 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N-terminal portion and / or C-terminal portion.Exemplary Pneumococcal Polypeptide Antigens: SP0435 Polypeptides

[0143] In some embodiments, an antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, a pneumococcal polypeptide antigen is or comprises a SP0435 polypeptide. SP0435 is a .S', pneumoniae protein. In some embodiments, an SP0435 polypeptide is an elongation factor P. In some embodiments, an SP0435 polypeptide is or comprises a full-length SP0435 polypeptide. For example, in some embodiments, a full-length SP0435 polypeptide has 186 amino acids (20 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 8. Without wishing to be bound by a particular theory, amino acids 1-61 of SEQ ID NO: 8 are predicted to be a dimerization domain of an SP0435 polypeptide (amino acids 1-61 of the full-length protein). Accordingly, in some embodiments, a SP0435 polypeptide may exclude such a dimerization domain, for example in some embodiments, to minimize or avoid the possibility of crosslinking or interference with a rhizavidin moiety. In some embodiments, an SP0435 polypeptide includes a portion of an SP0435 polypeptide (e.g., a portion of the SP0435 polypeptide of SEQ ID NO: 8, which portion includes 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 contiguous amino acids of SEQ ID NO: 8). In some embodiments, a portion of an SP0435 polypeptide corresponds to a protein having amino acids 62-185 of the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, an SP0435 polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and / or insertion) from a naturally- occurring wild-type SP0435 polypeptide sequence. For example, an SP0435 polypeptide may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to 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, 65, 70, 75, 80, 85, 90, 95, 100, 150, or more consecutive amino acids of the sequence shown in SEQ ID NO: 8). Alternatively, an SP0435 polypeptide may contain a portion (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) of a sequence that is at least 60% or more (e.g., at least 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 8. In some embodiments, a nucleotide sequence encoding an SP0435 polypeptide is provided herein as SEQ ID NO: 17. In some embodiments, an SP0434 polypeptide may comprises no more than 25 (including, 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., deletion, substitution, and / or insertion) within the sequence of SEQ ID NO: 8 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N-terminal portion and / or C-terminal portion. In some embodiments, an SP0435 polypeptide is one described in WO 2011 / 112906, the entire content of which is incorporated herein by reference for the purposes described herein.Exemplary Pneumococcal Polypeptide Antigens: SP0785 Polypeptides

[0144] In some embodiments, an antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, a pneumococcal polypeptide antigen is or comprises a SP0785 polypeptide. SP0785 is a conserved hypothetical S. pneumoniae protein, for example, in some embodiments as described in WO 2014 / 124228, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an SP0785 polypeptide is an efflux transporter protein conserved across 5. pneumoniae strains. In some embodiments, an SP0785 polypeptide is or comprises a full-length SP0785 polypeptide. For example, in some embodiments, a full-length SP0785 polypeptide has 399 amino acids (38 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 10. Without wishing to be bound by a particular theory, amino acids 1-32 of SEQ ID NO: 10 are predicted to be a signal sequence and transmembrane domain of an SP0785 polypeptide (amino acids 1-32 of the full-length protein). Accordingly, in some embodiments, an SP0785 polypeptide may exclude such a signal sequence and transmembrane domain. In some embodiments, an SP0785 polypeptide includes a portion of an SP0785 polypeptide (e.g., a portion of the SP0785 polypeptide of SEQ ID NO: 10, which portion includes 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 contiguous amino acids of SEQ ID NO: 10). In some embodiments, a portion of an SP0785 polypeptide corresponds to a protein having amino acids 33-399 of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments,an SP0785 polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and / or insertion) from a naturally-occurring wild-type SP0785 polypeptide sequence. For example, an SP0785 polypeptide may contain an amino acid sequence that is at least 60% or more e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 10 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, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or more consecutive amino acids of the sequence shown in SEQ ID NO: 10). Alternatively, an SP0785 polypeptide may contain a portion (e.g., at least 7, 8, 9, 10, 1 1 , 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 400 consecutive amino acids) of a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 10. In some embodiments, a nucleotide sequence encoding an SP0785 polypeptide is provided herein as SEQ ID NO: 18. In some embodiments, an SP0785 polypeptide may comprises no more than 25 (including, 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., deletion, substitution, and / or insertion) within the sequence of SEQ ID NO: 10 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N-terminal portion and / or C-terminal portion.Exemplary Pneumococcal Polypeptide Antigens: SP1500 Polypeptides

[0145] In some embodiments, an antigenic polypeptide described herein is or comprises a pneumococcal polypeptide antigen. In some embodiments, a pneumococcal polypeptide antigen is or comprises a SP1500 polypeptide. SP1500 is a S. pneumoniae protein, for example, in some embodiments as described in WO 2014 / 124228, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an SP1500 polypeptide is an Amino Acid ABC Transporter, amino acidbinding polypeptide conserved across 5. pneumoniae strains. In some embodiments, an SP1500 polypeptide is or comprises a full-length SP1500 polypeptide. For example, in some embodiments, a full-length SP1500 polypeptide has 278 amino acids (28 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 12. Without wishing to be bound by a particular theory, amino acids 1-26 of SEQ ID NO: 12 are predicted to be a signal sequence of an SP1500 polypeptide (amino acids 1-26 of the full-length protein).Accordingly, in some embodiments, a SP1500 polypeptide may exclude such a signal sequence. In some embodiments, an SP1500 polypeptide includes a portion of an SP1500 polypeptide (e.g., a portion of the SP1500 polypeptide of SEQ ID NO: 12, which portion includes 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 contiguous amino acids of SEQ ID NO: 12). In some embodiments, a portion of an SP1500 polypeptide corresponds to a protein having amino acids 27-278 of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, an SP1500 polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and / or insertion) from a naturally-occurring wild-type SP1500 polypeptide sequence. For example, an SP1500 polypeptide may contain an amino acid sequence that is 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%) identical to SEQ ID NO: 12 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, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or more consecutive amino acids of the sequence shown in SEQ ID NO: 12). Alternatively, an SP1500 polypeptide may contain a portion (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) of a sequence that is 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%) identical to SEQ ID NO: 12. In some embodiments, a nucleotide sequence encoding an SP1500 polypeptide is provided herein as SEQ ID NO: 19. In some embodiments, an SP1500 polypeptide may comprises no more than 25 (including, 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. , deletion, substitution, and / or insertion) within the sequence of SEQ ID NO: 12 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N-terminal portion and / or C-terminal portion.

[0146] In some embodiments, nucleic acid sequences encoding a pneumolysin polypeptide (SEQ ID NO: 4), an SP0435 polypeptide (SEQ ID NO: 8), SP0785 polypeptide (SEQ ID NO: 10), and an 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 degeneracy in the genetic code, those of ordinary skill in the art would understand that otherDNA sequences (including codon-optimized sequences) could encode these polypeptides, as well as the others disclosed herein.

[0147] In some embodiments, the present disclosure includes nucleic acid sequences encoding any of the amino acids described herein. Due to degeneracy in the genetic code, those of ordinary skill in the art would understand that other DNA sequences (including codon-optimized sequences) could encode these polypeptides, as well as the others disclosed herein.Fusion Proteins that Include Antigenic Polypeptides

[0148] Antigenic polypeptides described herein can be part of a fusion protein. For example, in some embodiments, an immunogenic complex described herein comprises a fusion protein that is or comprises a complementary affinity molecule and one or more antigenic polypeptides described herein. In some embodiments, a fusion protein comprises two antigenic polypeptides described herein. In some embodiments, a fusion protein comprises three antigenic polypeptides described herein. In some embodiments, a fusion protein comprises four antigenic polypeptides described herein. In some embodiments, a fusion protein comprises 6-10 antigenic polypeptides described herein. In some embodiments, a fusion protein comprises 11-15 antigenic polypeptides described herein. In some embodiments, a fusion protein comprises 15-20 antigenic polypeptides described herein.

[0149] In some embodiments, a fusion protein has carrier properties. In some embodiments, a fusion protein has antigenic properties. In some embodiments, a fusion protein has carrier properties and antigenic properties.

[0150] In some embodiments, a fusion protein comprises at least one antigenic polypeptide of, or derived from, a bacteria, a fungus, a parasite, a virus, or a cancer or tumor. In some embodiments, a fusion protein comprises an antigenic polypeptide that is or comprises a .S', pneumoniae polypeptide antigen (e.g., in some embodiments, a pneumolysin polypeptide antigen, a SP0435 polypeptide antigen, a SP0785 polypeptide antigen, or a SP1500 polypeptide antigen).

[0151] In some embodiments, a fusion protein comprises one or more antigenic polypeptides of S. pneumoniae having an amino acid sequence that is least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%. at least 98%, at least 99% identical toany of SEQ ID NOs: 4-13, or antigenic fragments thereof. In some embodiments, a fusion protein comprises one or more antigenic polypeptides of 5. pneumoniae having an amino acid sequence comprising any of SEQ ID NOs: 4-13, or antigenic fragments thereof. In some embodiments, a fusion protein comprises two antigenic polypeptides having an amino acid sequence that is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of SEQ ID NOs: 4-13, or antigenic fragments thereof. In some embodiments, a fusion protein comprises two antigenic polypeptides having an amino acid sequence comprising any of SEQ ID NOs: 4-13, or antigenic fragments thereof. In some embodiments, a fusion protein as described herein further comprises a biotin-binding moiety, for example, in some embodiments, a biotin-binding moiety that is or comprises a rhizavidin polypeptide having an amino acid sequence that is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or biotin-binding fragments thereof. In some embodiments, a fusion protein as described herein further comprises a biotin-binding moiety, for example, in some embodiments, a biotin-binding moiety that is or comprises a rhizavidin polypeptide having the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or biotin-binding fragments thereof.

[0152] In some embodiments, a fusion protein comprises a pneumolysin polypeptide as described herein. For example, in some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of 5. pneumoniae PdT(G294P) polypeptide), or an antigenic fragment thereof. In some embodiments, a pneumolysin polypeptide described herein may be truncated from its N-terminal portion and / or C-terminal portion. In some embodiments, a fusion protein as described herein further comprises a rhizavidin polypeptide, for example, in some embodiments, a rhizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3, or biotin-binding fragments thereof.

[0153] In some embodiments, a fusion protein comprises an SP0435 polypeptide. For example, in some embodiments, a fusion protein comprises a polypeptide comprising 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 to thesequence of SEQ ID NO: 9 (amino acids 62-185 of 5. pneumoniae SP0435 polypeptide), or an antigenic fragment thereof. In some embodiments, an SP0435 polypeptide described herein may be truncated from its N-terminal portion and / or C-terminal portion. In some embodiments, a fusion protein as described herein further comprises a rhizavidin polypeptide, for example, in some embodiments, a rhizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3, or biotin-binding fragments thereof.

[0154] In some embodiments, a fusion protein comprises an SP0785 polypeptide. For example, in some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of S. pneumoniae SP0785 polypeptide), or an antigenic fragment thereof. In some embodiments, an SP0785 polypeptide described herein may be truncated from its N-terminal portion and / or C-terminal portion. In some embodiments, a fusion protein as described herein further comprises a rhizavidin polypeptide, for example, in some embodiments, a rhizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3, or biotin-binding fragments thereof.

[0155] In some embodiments, a fusion protein comprises an SP1500 polypeptide. For example, in some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S. pneumoniae SP1500 polypeptide), or an antigenic fragment thereof. In some embodiments, an SP1500 polypeptide described herein may be truncated from its N-terminal portion and / or C-terminal portion. In some embodiments, a fusion protein as described herein further comprises a rhizavidin polypeptide, for example, in some embodiments, a rhizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3, or biotin-binding fragments thereof.

[0156] In some embodiments, a fusion protein comprises one or more polypeptides homologous to a polypeptide described herein (e.g., an antigenic polypeptide isolated from different serotypes, strains, or species). Individual serotypes, strains, or species can contain numerous mutations relative to each other, and some of these can result in different protein sequences between the different serotypes, strains, or species. In some embodiments, a fusion protein comprises one or more polypeptides homologous to a S. pneumoniaepolypeptide described herein, e.g., a Ply polypeptide, an SP0435 polypeptide, an SP0785 polypeptide or an SP1500 polypeptide isolated from different serotypes of 5. pneumoniae. In some embodiments, antigenic polypeptides have 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 to the polypeptides of any of SEQ ID NOs: 4-13, or antigenic fragments thereof. Serotype, strain, or species variation may be used to design such variants of the antigenic polypeptides described herein.

[0157] In some embodiments, fusion proteins described herein comprise one or more fragments of one or more polypeptides described herein, e.g., biotin-binding fragments of rhizavidin or antigenic fragments of an antigenic polypeptide (e.g., a Ply polypeptide or antigenic fragments thereof, an SP0435 polypeptide or antigenic fragments thereof, a SP0785 polypeptide or antigenic fragments thereof (e.g. , with or without a signal sequence), or a SP1500 polypeptide or antigenic fragments thereof (e.g., with or without a signal sequence)). In some embodiments, a fusion protein described herein comprises a truncated version of a polypeptide described herein, which truncated version is close in size to a polypeptide having the amino acid sequences of any of SEQ ID NOs: 4-13. For example, in some embodiments, such a truncated polypeptide may lack at most one, two, three, four, five, ten, or twenty amino acids from one or both termini from its parent polypeptide. In some embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus. In some embodiments, a truncated polypeptide has an amino acid sequence that is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of SEQ ID NOs: 4-13 lacking 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both. In some embodiments, a truncated polypeptide has the amino acid sequence of any of SEQ ID NOs: 4-13 lacking 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both. In some embodiments, a truncated polypeptide has an amino acid sequence that is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of SEQ ID NOs: 4-13 lacking 1-10 acid residues from the N-terminus, C-terminus, or both. In some embodiments, a truncated polypeptide has the amino acid sequence of any of SEQ ID NOs: 4-13 lacking 1-10 amino acid residues from the N-terminus, C-terminus, or both. For instance, a truncated polypeptide may lack 10 amino acid residues at both the N-terminus and C-terminus of any one of SEQ ID NOs: 4-13, resulting in a protein lacking 20 amino acidresidues. Internal deletions, e.g. , of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.

[0158] In some embodiments, a fusion protein comprises an N-terminal polypeptide and a C-terminal polypeptide. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide is an antigenic polypeptide described herein (e.g., a polypeptide having an amino acid sequence comprising one or more of SEQ ID NOs: 4-13), or an antigenic fragment or variant thereof. In some embodiments, one or both of the N- terminal polypeptide and the C-terminal polypeptide is a biotin-binding moiety, for example a polypeptide having an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a biotin-binding fragment thereof. In some embodiments, one of the N- terminal polypeptide or the C-terminal polypeptide is a biotin-binding moiety, e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a biotin-binding fragment thereof, 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 NOs: 4-13), or an antigenic fragment or variant thereof.

[0159] In some embodiments, an N-terminal polypeptide and a C-terminal polypeptide present in a fusion protein are directly bound to each other. In some embodiments, an N-terminal polypeptide and a C-terminal polypeptide present in a fusion protein are linked via a linker peptide (e.g., a linker disclosed herein). The length and / or amino acids of a linker, when present, can be adjusted to obtain a more flexible, semi-rigid, or rigid linker. In some embodiments, a linker can be a GS-enriched linker. In some embodiments, a linker can be an A-enriched linker. A linker can generally be from 1-40, such as 3-10 or 10-30 and specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Exemplary flexible peptide linkers are shown as SEQ ID NOs: 38-42 and 45-47.

[0160] In some embodiments, a fusion protein comprises one or more linkers described herein. In some embodiments, a fusion protein comprises at least one linker. In some embodiments, a fusion protein comprises at least two linkers. In some embodiments, a linker comprises a polypeptide comprising an amino acid sequence of GGGGSSS (SEQ ID NO: 38). In some embodiments, a linker comprises a polypeptide comprising an amino acid sequence of AAA. In some embodiments, a linker comprises a polypeptide comprising an amino acid sequence of AAAA (SEQ ID NO: 66). In some embodiments, a linker comprisesa polypeptide comprising an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 41). In some embodiments, a fusion protein comprises a first linker having the amino acid sequence of GGGGSSS (SEQ ID NO: 38) and a second linker having the amino acid sequence of AAA. In some embodiments, a linker sequence may be a residual amino acid sequence from a restriction site. For example, in some embodiments, a fusion protein comprises an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, a fusion protein comprises a linker of GGGGSSS (SEQ ID NO: 38) and an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, the one or more linkers are selected from GGGGSSS (SEQ ID NO: 38) and AAAA (SEQ ID NO: 66). In some embodiments, a fusion protein comprises a linker of GGGGSSS (SEQ ID NO: 38) and AAAA (SEQ ID NO: 66).

[0161] In some embodiments, a fusion protein described herein comprises a biotinbinding moiety. In some embodiments, the fusion protein 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 polypeptide antigens. In some embodiments, the biotin-binding moiety of a fusion protein comprises rhizavidin or a biotin-binding fragment thereof, which in some embodiments as further described in WO 2012 / 155053, the contents of which are herein incorporated by reference in their entirety for the purposes described herein.

[0162] In some embodiments, a fusion protein described herein comprises a biotinbinding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof. In some embodiments, a fusion protein comprises a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin, denoted Rhavi) or SEQ ID NO: 3, or biotin-binding fragment thereof.

[0163] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P) polypeptide), or an antigenic fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of S. pneumoniae SP0435 polypeptide) or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, such one or more linkers are independently selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs: 38-67 and AAA. In some embodiments, such one or more linkers are each GGGGSSS (SEQ ID NO: 38).

[0164] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of S. pneumoniae SP0785 polypeptide), or an antigenic fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S. pneumoniae SP1500 polypeptide) or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, such one or more linkers are independently selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs: 38-67 and AAA. In some embodiments, such one or more linkers are selected from GGGGSSS (SEQ ID NO: 30) and AAA. In some embodiments, the fusion protein comprises an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of GGGGSSS (SEQ ID NO: 38) and an amino acid sequence AAA residual from a Not I restriction site.

[0165] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin, denoted Rhavi) or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of .S', pneumoniae PdT(G294P) polypeptide) or an antigenic fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of S. pneumoniae SP0435 polypeptide) or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, such one or more linkers are independently selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs: 38-67 and AAA. In some embodiments, such one or more linkers are each GGGGSSS (SEQ ID NO: 38). In some embodiments, a 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 to 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.

[0166] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin, denoted Rhavi) or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of 5. pneumoniae SP0785 polypeptide) or an antigenic fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of .S'. pneumoniae SP1500 polypeptide) or an antigenicfragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, such one or more linkers are independently selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs: 38-67 and AAA. In some embodiments, such one or more linkers are selected from GGGGSSS (SEQ ID NO: 38) and AAA. In some embodiments, the fusion protein comprises an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of GGGGSSS (SEQ ID NO: 38) and an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, a 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 to the sequence SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO: 30 (CPI) or SEQ ID NO: 31.

[0167] In some embodiments, the present disclosure provides fusion proteins 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% sequence identity to a fusion protein having an amino acid sequence of any one of SEQ ID NOs: 20-31. In some embodiments, a fusion protein is or includes 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 to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a fusion protein is or includes 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 to SPP2. In some embodiments, a fusion protein is or includes 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 to SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, a fusion protein is or includes 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 to CPI.

[0168] In some embodiments, a fusion protein described herein comprises an antigenic fragment of a fusion protein having an amino acid sequence of any one of SEQ ID NOs: 20-31. For example, a fusion protein may lack at most one, two three, four, five, ten, ortwenty amino acids from the N-terminus, C-terminus, or both, of any one of SEQ ID NOs: 20-31. In some embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus. In some embodiments, a fusion protein is or includes an antigenic fragment of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a fusion protein is or includes an antigenic fragment of SPP2. In some embodiments, a fusion protein is or includes an antigenic fragment of SEQ ID NO: 30 or SEQ ID NO: 1. In some embodiments, a fusion protein is or includes an antigenic fragment of CPI.

[0169] In some embodiments, a fusion protein described herein includes a variant or fragment of a polypeptide having an amino acid sequence of SEQ ID NOs: 1-13. In some embodiments, a fusion protein described herein includes a polypeptide encoded by a variant or fragment of a gene having a nucleic acid sequence of SEQ ID NOs: 1-13. In some embodiments, a fragment included in a fusion protein described herein is close in size to a full-length polypeptide or a polypeptide having an amino acid sequence of SEQ ID NOs: 1- 13. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini. In some embodiments, the fragment is 25-50 amino acids in length, or 50-100, or 100-150, or 150-200, or 200-250, or 250-300, or 300-350 amino acids in length. In some embodiments, the fragments result from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g. , the baculovirus expression system), a yeast (e.g., S. cerevisiae or .S', pombe) cell line, or a mammalian (e.g., human or Chinese Hamster Ovary) cell line. The fragments described above or sub-fragments thereof (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 IL- 17 released by at least 1.5-fold or 2-fold or more (e.g., either as an absolute measure or relative to a control protein).

[0170] In some embodiments, fusion proteins described herein can be useful in immunogenic complexes described herein.Linkers or Spacers

[0171] In some embodiments, a polypeptide described herein is coupled to one or more entities of interest (e.g., small molecules, polypeptides, polynucleotides, lipids, polysaccharides, etc.) with one or more linkers. In some embodiments, a fusion protein described herein comprises one or more linkers. In some embodiments, a linker is or comprises one or more amino acids. In some embodiments, a fusion protein comprises an antigenic polypeptide joined to a biotin-binding moiety by a linker. In some embodiments, a fusion protein comprises a first antigenic polypeptide, a second antigenic polypeptide, a biotin-binding moiety, and at least one linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are joined by a linker. In some embodiments, the first antigenic polypeptide or the second antigenic polypeptide are joined to the biotin-binding moiety by a linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are joined by a first linker; and the first antigenic polypeptide or the second antigenic polypeptide are joined to the biotin-binding moiety by a second linker.

[0172] In some embodiments, a fusion protein described herein comprises a first linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38), and a second linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38). In some embodiments, a fusion protein described herein comprises a first linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38), and a second linker comprising the amino acid sequence of AAA.

[0173] In some embodiments, a linker interposes a structure between two protein moieties. In some embodiments, the structure is or comprises an a-helix. In some embodiments, the structure is or comprises a 0-strand. In some embodiments, the structure is or comprises a coil / bend. In some embodiments, the structure is or comprises a turn. In some embodiments, a linker decreases steric hindrance between two protein moieties joined by the linker. In some embodiments, a linker decreases unfavorable interactions between two protein moieties joined by the linker. In some embodiments, a linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline, and / or alanine residues. In some embodiment, a linker is hydrophilic. In some embodiments, a linker is hydrophobic. In some embodiments, a linker increases the stability of a fusion protein containing the linker.

[0174] In some embodiments, a linker does not interfere with the folding or activity of a polypeptide of interest (e.g., in some embodiments, an antigenic polypeptide) to which it is joined. In some embodiments, a linker does not interfere with the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments, a linker does not reduce the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments, a linker does not eliminate the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments, the effect of the linker is determined by comparing the polypeptide with the polypeptide joined to the linker.

[0175] In some embodiments, a linker does not interfere with the folding of a biotinbinding moiety to which it is joined. In some embodiments, a linker does not interfere with the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments, a linker does not reduce the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments, a linker does not eliminate the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments, the effect of the linker is determined by comparing the biotin-binding moiety with the biotin-binding moiety joined to the linker.

[0176] In some embodiments, a linker is not antigenic. In some embodiments, a linker does not elicit a T cell response. In some embodiments, a linker does not elicit a B cell response. In some embodiments, a linker does not induce a T cell or a B cell response.

[0177] In some embodiments, a linker comprises two or more amino acids. In some embodiments, a 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 length. In some embodiments, a linker comprises between 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, a linker is or comprises a peptidyl 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.

[0178] In some embodiments, a linker is a flexible linker. Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar e.g. , Gly) or polar e.g. , Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forminghydrogen bonds with water molecules, and therefore reduce unfavorable interactions between the linker and the protein moieties. In some embodiments, a linker comprises small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. In some embodiments, a linker is a Gly-Ser linker.

[0179] In some embodiments, a linker is or comprises an amino acid sequence of GGGGSSS (SEQ ID NO: 38). In some embodiments, a linker is or comprises a sequence of (GGGGS)n (SEQ ID NO: 39), where n represents the number of repeating GGGGS (SEQ ID NO: 40) units and is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a polypeptide linker may have an amino acid sequence that is or comprises GGGGSGGGGSGGGGS (SEQ ID NO: 41) (z.e., (GGGGS)3) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) (i.e., (GGGGS)6). In some embodiments, a linker comprises one or more of Gly, Ser, Thr, Ala, Lys, and Glu. In some embodiments, a linker is or comprises KESGSVSSEQLAQFRSLD (SEQ ID NO: 43). In some embodiments, a linker is or comprises EGKSSGSGSESKST (SEQ ID NO: 44). In some embodiments, a linker is or comprises (Gly)n(SEQ ID NO: 45) 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, a linker is or comprises GGG. In some embodiments, a linker is or comprises (Gly)e (SEQ ID NO: 46). In some embodiments, a linker is or comprises (Gly)s (SEQ ID NO: 47). In some embodiments, a linker is or comprises GSAGSAAGSGEF (SEQ ID NO: 48). In some embodiments, a linker is or comprises an amino acid sequence AAA. In some embodiments, such a linker may be synthesized, or derived from amino acid residues from a restriction site (e.g., a Not I restriction site).

[0180] In some embodiments, a linker is a rigid linker. Rigid linkers are useful to keep a fixed distance between domains and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the fusion. In some embodiments, a linker is or comprises (EAAAK)n(SEQ ID NO: 49) where n represents the number of repeating EAAAK (SEQ ID NO: 50) 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, a linker is or comprises A(EAAAK)nA, (SEQ ID NO: 51) where n represents the number of repeating EAAAK (SEQ ID NO: 50) 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, a linker is or comprises A(EAAAK)nA (SEQ ID NO: 52),where n represents the number of repeating EAAAK (SEQ ID NO: 50) units and is 2, 3, 4, or 5. In some embodiments, a linker is or comprises A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 53). In some embodiments, a linker is or comprises [A(EAAAK)nA]m, (SEQ ID NO: 54) wherein n is 2, 3, or 4 and m is 1 or 2. In some embodiments, a linker is or comprises AEAAAKEAAAKA (SEQ ID NO: 55).

[0181] In some embodiments, a linker is or comprises (X-Pro)n(SEQ ID NO: 56), with X designating any amino acid, where n represents the number of repeating X-Pro units and is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises (Ala-Pro)n(SEQ ID NO: 57), 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, a linker is or comprises (Ala-Pro)n(SEQ ID NO: 58), 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.

[0182] In some embodiments, a linker is or comprises (Lys-Pro)n(SEQ ID NO: 59), 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, a linker is or comprises (Glu-Pro)„ (SEQ ID NO: 60), 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, a linker is or comprises (Ala- Pro)? (SEQ ID NO: 61).

[0183] In some embodiments, a linker is or comprises GAPGGGGGAAAAAGGGGGGAP (GAG linker, SEQ ID NO: 62). In some embodiments, a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 linker, SEQ ID NO: 63). In some embodiments, a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGG GGGAP (GAG3 linker, SEQ ID NO: 64).

[0184] In some embodiments, a linker is or comprises VSDP (SEQ ID NO: 65).

[0185] In some embodiments, a linker is or comprises AAAA (SEQ ID NO: 66).

[0186] In some embodiments, a linker is or comprises GGGG (SEQ ID NO: 67).

[0187] Suitable linkers or spacers also include those having an amino acid sequence 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 homologous or identical to theabove exemplary linkers. In some embodiments, the linker comprises a polypeptide comprising 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%, or 100% identity to the sequence of GGGGSSS (SEQ ID NO: 38).

[0188] Additional linkers suitable for use with some embodiments may be found in U.S. Patent Publication No. 2012 / 0232021, filed on March 2, 2012, and [Chen, 2013] the disclosures of which is hereby incorporated by reference in their entireties.Tags

[0189] In some embodiments, a polypeptide (e.g., a fusion protein) described herein may comprise one or more tags. In some embodiments, a fusion protein described herein comprises one or more tags. A tag may be present at the N-terminal or C-terminal of a polypeptide. For instance, tags may be added to a polypeptide (e.g., via additions or modifications on the encoding DNA sequence) to facilitate purification, detection, solubility, or confer other desirable characteristics on the polypeptide. In some embodiments, a tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification. In some embodiments, a tag is, comprises, or is derived from one or more of poly-histidine (His), Glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose binding protein (MBP), vesicular Stomatitis viral glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), calmodulin, Nus, S tags, lipoprotein D, and galactosidase. In some embodiments, a His tag is or comprises an amino acid sequence of Hn, wherein n is an integer between 2 and 10 (SEQ ID NO: 68). Exemplary His tags include HHHHHH (SEQ ID NO: 69) and MSYYHHHHHH (SEQ ID NO: 70). In other embodiments, a polypeptide described herein is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag. In some embodiments, a fusion protein described herein comprises (i) a polypeptide of amino acid sequence of any of SEQ ID NOs: 4-13 or fusion protein of amino acid sequence of any of SEQ ID NOs: 20-31, and (ii) a tag having no more than 1, 2, 3, 4, 5, 10, or 20 amino acids on one or both termini of the polypeptide or fusion protein of (i).

[0190] In some embodiments, a polypeptide described herein may contain a membrane translocating sequence (MTS), for example, in some embodiments, to facilitate introduction of the fusion protein into a mammalian cell and subsequent stimulation of thecell-mediated immune response. Exemplary membrane translocating sequences include, but are not limited to the hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of a synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin 3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in U.S. Patent Nos. 6,248,558; 6,432,680; and 6,248,558.

[0191] In some embodiments, a polypeptide (e.g., a fusion protein) described herein may comprise one or more tags. In some embodiments, a fusion protein comprises one or more tags. A tag may be N-terminal or C-terminal. For instance, tags may be added to a polypeptide (via additions or modifications on the encoding DNA sequence) to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein. In some embodiments, a tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification. In some embodiments, a tag is, comprises, 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 viral glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), Calmodulin, Nus, S tags, lipoprotein D, and galactosidase. In some embodiments, a His tag is or comprises an amino acid sequence of Hn, wherein n is an integer between 2 and 10 (SEQ ID NO: 68). Exemplary His tags include HHHHHH (SEQ ID NO: 69) and MSYYHHHHHH (SEQ ID NO: 70). In other embodiments, a fusion protein described herein is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag. In some embodiments, a fusion protein described herein comprises (i) a polypeptide of amino acid sequence of any of SEQ ID NOs: 1-13 or fusion protein of amino acid sequence of any of SEQ ID NOs: 20-31, and (ii) a tag having no more than 1, 2, 3, 4, 5, 10, or 20 amino acids on one or both termini of the polypeptide or fusion protein of (i).

[0192] In some embodiments, a polypeptide described herein may contain a membrane translocating sequence (MTS), for example, in some embodiments, to facilitate introduction of the fusion protein into a mammalian cell and subsequent stimulation of the cell-mediated immune response. Exemplary membrane translocating sequences include, but are not limited to the hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of a synuclein, the third helix of the Antennapedia homeodomain, SN50,integrin 3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in U.S. Patent Nos. 6,248,558; 6,432,680; and 6,248,558.Signal Peptides

[0193] In some embodiments, a polypeptide described herein comprises a signal peptide. In some embodiments, a fusion protein comprises a signal peptide. Signal peptides, which may also be called leader peptides, function in translocation of polypeptides through a cellular membrane (e.g., the plasma membrane, endoplasmic reticulum, etc.). In some embodiments, a polypeptide comprises a signal peptide at or near its N-terminus. In some embodiments, the signal sequence may or may not be cleaved off (e.g., by a signal peptidase) after translocation through a membrane.

[0194] A signal sequence can be directly linked e.g. , by a peptide bond) or indirectly linked e.g., by a linker) to a polypeptide. In some embodiments, the signal sequence can be linked to a polypeptide by a peptide linker disclosed herein.

[0195] In some embodiments, a signal peptide may be a bacterial signal peptide, a viral signal peptide, or eukaryotic signal peptide. In some embodiments, a eukaryotic signal peptide is a mammalian signal peptide (e.g. , a signal peptide from, or derived from, a human protein).

[0196] In some embodiments, a signal peptide may be a bacterial signal peptide. Bacterial signal peptides may be used to promote translocation of a polypeptide into periplasmic space of bacteria. In some embodiments, the signal peptide is cleaved off from the polypeptide after translocation into the periplasmic space of a bacterium (e.g., E. coli). Signal peptides that direct secretion of proteins from bacterial cells are well known in the art, for example as disclosed in International application WO 2005 / 071088.

[0197] Any signal peptide known to one of ordinary skill in the art can be used. In some embodiments, a signal peptide is or comprises the amino acid sequence: MKKIWLALAGLVLAFSASA (SEQ ID NO: 71), MAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 72), MKKVAAFVALSLLMAGC (SEQ ID NO: 73), MKKIMLVITLILVSPIAQQTEAKD (SEQ ID NO: 74), MKKKIISAILMSTVILSAAAPLSGVYADT (SEQ ID NO: 75), MKKRKVLIPLMALSTILVSSTGNLEVIQAEV (SEQ ID NO: 76), MNMKKATIAATAGIAVTAFAAPTIASAST (SEQ ID NO: 77),MQKTRKERILEALQEEKKNKKSKKFKTGATIAGVTAIATSITVPGIEVIVSADE (SEQ ID NO: 78), MKKLKMASCALVAGLMFSGLTPNAFAED (SEQ ID NO: 79), MAKKFNYKLPSMVALTLVGSAVTAHQVQAAE (SEQ ID NO: 80), MTDKKSENQTEKTETKENKGMTRREMLKLSAVAGTGIAVGATGLGTILNVVDQVD KALT (SEQ ID NO: 81), MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLGLGLTIAQSVGAFG (SEQ ID NO: 82) or a derivative or functional portion thereof. In some embodiments, a signal peptide is or comprises the amino acid sequence of a human Ig heavy chain V-III region VH26 signal peptide or a derivative or functional portion thereof. In some embodiments, a signal peptide is or comprises the amino acid sequence of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 83) or a derivative or functional portion thereof. In some embodiments, a signal peptide is or comprises a human IgG2 heavy chain signal peptide, or a derivative or functional portion thereof. In some embodiments, a signal peptide is or comprises the amino acid sequence ofMGWSCIILFLVATATGVHS (SEQ ID NO: 84) or a derivative or functional portion thereof.

[0198] Other examples of signal peptides can be found at Signal Peptide Website, a signal peptide database, which is found at www.signalpeptide.de.

[0199] In some embodiments, a polypeptide described herein lacks a signal peptide.Affinity Molecule Pairs

[0200] As described herein, immunogenic complexes of the present disclosure include 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 that is complementary to the first affinity molecule). In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of biotin / biotin- binding moiety, antibody / antigen, enzyme / substrate, receptor / ligand, metal / metal-binding protein, carbohydrate / carbohydrate binding protein, lipid / lipid-binding protein, and His tag / His tag-binding molecule. In some embodiments, a first affinity molecule is associated with a polymer (e.g., but not limited to a polysaccharide). In some embodiments a second affinity molecule is associated with a polypeptide of interest (e.g., but not limited to a polypeptide antigen).

[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, e.g., a biotin-binding protein or polypeptide, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, a fusion protein of an immunogenic complex comprises a biotin-binding moiety, and one or more polypeptide antigens. In some embodiments, a fusion protein comprises a biotin-binding moiety and two or more polypeptide antigens. In some embodiments, the biotin-binding moiety is a biotin-binding moiety that can form a dimer. In some embodiments, the hiotin-binding moiety is a biotin-binding moiety that can form a tetramer. In some embodiments, the biotin-binding moiety is or comprises rhizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrAvidin, CaptAvidin™, or a biotin-binding domain or biotin-binding fragment thereof, or a combination thereof. In some embodiments, the biotin-binding moiety is or comprises rhizavidin, or a biotin-binding domain or biotin-binding fragment thereof.

[0202] In some embodiments, MAPS complexes disclosed herein utilize the high affinity (dissociation constant [KD] ~ 10"15M) non-covalent binding between biotin and rhizavidin, a biotin-binding protein that has no significant predicted homology with human proteins. Rhizavidin, a naturally occurring dimeric protein in the avidin protein family, was first discovered in Rhizobium etli, a symbiotic bacterium of the common bean. Rhizavidin has only a 22% amino acid identity with chicken avidin, a protein commonly found in eggs, but with high conservation of amino acid residues involved in biotin binding. No crossreactivity to rhizavidin is observed in human serum samples obtained from subjects exposed to avidin | Helppolainen et al, 20071, suggesting that rhizavidin antibodies may not cross-react with chicken avidin. Biotin conjugates have been used in several clinical applications without any reported adverse events [Buller et al, 2014; Paty et al, 2010; Lazzeri et al, 2004].

[0203] In some embodiments, a biotin-binding moiety that is useful in accordance with the present disclosure comprises rhizavidin or a biotin-binding domain or biotin-binding fragment thereof, for example, in some embodiments as further described in WO 2012 / 155053, the contents of which are herein incorporated by reference in their entirety for the purposes described herein. In some embodiments, a biotin-binding moiety 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 to rhizavidin, or a biotinbinding domain or biotin-binding fragment thereof. In some embodiments, a biotin-bindingmoiety that is useful in accordance with the present disclosure comprises a polypeptide of SEQ ID NO: 1 or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, a biotin-binding moiety 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%, 100% identity to the sequence of SEQ ID NO: 1, or biotin-binding domain or biotin-binding fragment thereof. In some embodiments, a biotin-binding moiety that is useful in accordance with the present disclosure comprises a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, a biotinbinding moiety 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 to the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or biotin-binding domain or biotin-binding fragment thereof.Fusion Protein SPP2

[0204] In some embodiments, a fusion protein described herein is or comprises a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein), and one or more polypeptides of or derived from .S', pneumoniae. In some embodiments, a provided fusion protein is SPP2, further described in International Application No. PCT / US2022 / 043156 filed on September 9, 2022, the contents of which are incorporated herein by reference in their entirety for the purposes described herein. In some embodiments, a fusion protein comprises (i) a biotin-binding moiety (e.g., a biotin-binding protein), (ii) a non-hemolytic pneumolysin polypeptide comprising mutations at amino acid residues 294, 385, 428, and 433 of wild-type Streptococcus pneumoniae pneumolysin or an antigenic fragment thereof; and (iii) a S. pneumoniae elongation factor P (SP0435) polypeptide or an antigenic fragment thereof. In some embodiments, a biotin-binding moiety is or comprises a rhizavidin polypeptide or a biotin-binding portion thereof. In some embodiments, a fusion protein comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein) (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof, and (iii) a polypeptide comprising an amino acid sequence having at least80%, 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of .S’, pneumoniae SP0435) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a fusion protein is or comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety 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 to pneumolysin 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 to SP0435 or an antigenic variant or fragment thereof. In some embodiments, a fusion protein is or comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety 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 to a non-hemolytic pneumolysin comprising mutations at amino acid residues 294, 385, 428, and 433 of wild-type S. pneumoniae pneumolysin 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 to SP0435 or an antigenic variant or fragment thereof.

[0205] In some embodiments, a fusion protein described herein comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein) and (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein) and (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of .S'. pneumoniae SP0435) or an antigenic variant or fragment thereof.

[0206] In some embodiments, a fusion protein described herein comprises a biotinbinding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof. In some embodiments, a fusion protein comprises a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotin-binding fragment thereof. In some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof. In some embodiments, the fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of S. pneumoniae SP0435) or an antigenic variant or fragment thereof.

[0207] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of .S'. pneumoniae SP0435) or an antigenic variant or fragment thereof. In some embodiments, such a fusion protein further comprises one or more linkers.

[0208] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of .S', pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of S. pneumoniae SP0435) or an antigenic variant or fragment thereof. In some embodiments, such a fusion protein further comprises one or more linkers.

[0209] In some embodiments, a fusion protein described herein is or comprises a SPP2 fusion protein. In some embodiments, a SPP2 fusion protein comprises, from N- terminus to C-terminus, (i) a biotin-binding moiety (<?.g., a biotin-binding protein), (ii) a first peptide linker, (iii) a pneumolysin (Ply) polypeptide described herein or an antigenic variant or fragment thereof; (iv) a second peptide linker, and (v) an SP0435 polypeptide described herein or an antigenic variant or fragment thereof. In some such embodiments, a biotinbinding moiety is or comprises rhizavidin or a biotin-binding portion thereof. In some embodiments, a SPP2 fusion protein comprises (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin) or to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) an pneumolysin (Ply) polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); and (v) an SP0435 polypeptide described herein. In some embodiments, a SPP2 fusion protein comprises, from N-terminus to C-terminus, (i) a biotin-binding moiety that is or comprises the polypeptide of SEQ ID NO: 1 (rhizavidin), SEQ ID NO: 2 (amino acids 45-179 of rhizavidin), or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) an pneumolysin (Ply) polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); and (v) an SP0435 polypeptide described herein. In some embodiments, such a SPP2fusion protein may further comprise a detection or purification tag, e.g. , a His tag. In some embodiments, a SPP2 fusion protein comprising a His tag is referred to as SPP2-H. In some such embodiments, a SPP2 fusion protein comprises an SP0435 polypeptide (e.g., ones described herein) between a biotin-binding moiety and a Ply polypeptide (e.g., ones described herein). In some embodiments, a SPP2 fusion protein may comprise a Ply polypeptide between a biotin- binding moiety and a SP0435 polypeptide. In some embodiments, a Ply polypeptide included in a fusion protein described herein 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 to the sequence of SEQ ID NO: 7 (amino acids 2-470 of S. pneumoniae PdT(G294P)) or an antigenic variant or fragment thereof. In some embodiments, an SP0435 polypeptide included in a fusion protein described herein 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 to the sequence of SEQ ID NO: 9 (amino acids 62-185 of .S', pneumoniae SP0435) or an antigenic variant or fragment thereof. In some embodiments, a 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 to the sequence SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a fusion protein described herein comprises the amino acid sequence SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a fusion protein described herein consists of the amino acid sequence SEQ ID NO: 20 or SEQ ID NO: 21.Fusion Protein CPI

[0210] In some embodiments, a fusion protein described herein is or comprises a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein), and one or more polypeptides of or derived from .S', pneumoniae. In some embodiments, a fusion protein described herein is CPI, further described in the International Patent Publication No. WO 2020 / 056127, the contents of which are incorporated herein by reference in their entirety for the purposes described herein. In some embodiments, a fusion protein comprises (i) a complementary affinity molecule described herein (e.g., a biotinbinding moiety described herein), (ii) a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 11 (amino acids 33-399 of S. pneumoniae SP0785) or an antigenic variant or fragment thereof, and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of 5. pneumoniae SP1500) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, a fusion protein is or comprises (i) a complementary affinity molecule described herein (e.g., a biotin- binding moiety 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 to 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%, or 100% identity to SP1500 or an antigenic variant or fragment thereof.

[0211] In some embodiments, a fusion protein described herein comprises (i) a complementary affinity molecule described herein (e.g. , a biotin-binding moiety described herein) and (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of S. pneumoniae SP0785) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein comprises (i) a complementary affinity molecule described herein e.g., a biotin-binding moiety described herein) and (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S. pneumoniae SP1500) or an antigenic variant or fragment thereof.

[0212] In some embodiments, a fusion protein described herein comprises a biotinbinding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof. In someembodiments, a fusion protein comprises a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotin-binding fragment thereof. In some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 1 1 (amino acids 33-399 of S. pneumoniae SP0785) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein comprises a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S. pneumoniae SP1500) or an antigenic variant or fragment thereof.

[0213] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof; (ii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of 5. pneumoniae SP0785) or an antigenic variant or fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S. pneumoniae SP1500) or an antigenic variant or fragment thereof. In some embodiments, such a fusion protein further comprises one or more linkers.

[0214] In some embodiments, a fusion protein described herein comprises each of: (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 11 (amino acids 33-399 of 5. pneumoniae SP0785) or an antigenic variant or fragment thereof; and (iii) a polypeptide comprising 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of 5. pneumoniae SP1500) or an antigenic variant or fragment thereof. In some embodiments, such a fusion protein further comprises one or more linkers.

[0215] In some embodiments, a fusion protein described herein is or comprises a CPI fusion protein. In some embodiments, a CPI fusion protein comprises, from N-terminus to C-terminus, (i) a biotin-binding moiety (e.g.. a biotin-binding protein), (ii) a first peptide linker, (iii) an SP1500 polypeptide described herein or an antigenic variant or fragment thereof; (iv) a second peptide linker, and (v) an SP0785 polypeptide described herein or an antigenic variant or fragment thereof. In some such embodiments, a biotin-binding moiety is or comprises rhizavidin or a biotin-binding portion thereof. In some embodiments, a CPI fusion protein comprises (i) a biotin-binding moiety that 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 to the sequence of SEQ ID NO: 1 (rhizavidin) or to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin) or SEQ ID NO: 3, or biotinbinding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) an SP0785 polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence of AAA; and (v) an SP1500 polypeptide described herein. In some embodiments, a CPI fusion protein comprises, from N-terminus to C-terminus, (i) a biotin-binding moiety that is or comprises the polypeptide of SEQ ID NO: 1 (rhizavidin), SEQ ID NO: 2 (amino acids 45-179 of rhizavidin), or SEQ ID NO: 3, or biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) an SP1500 polypeptide described herein; (iv) a second polypeptide linker comprising the amino acid sequence of AAA; and (v) an SP0785 polypeptide described herein. In some embodiments, such a CPI fusion protein may further comprise a detection or purification tag, e.g., a His tag. In some such embodiments, a CPI fusion protein comprises an SP1500 polypeptide between a biotin-binding moiety and an SP0785 polypeptide. In some embodiments, a CPI fusion protein may comprise an SP0785 polypeptide between a biotin-binding moiety and a SP1500 polypeptide. In some embodiments, an SP0785 polypeptide included in a fusion protein described herein is orcomprises 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 to the sequence of SEQ ID NO: 11 (amino acids 33-399 of S. pneumoniae SP0785) or an antigenic variant or fragment thereof. In some embodiments, an SP1500 polypeptide included in a fusion protein described herein 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 to the sequence of SEQ ID NO: 13 (amino acids 27-278 of S', pneumoniae SP1500) or an antigenic variant or fragment thereof. In some embodiments, a fusion protein 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 to the sequence SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, a fusion protein comprises the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, a fusion protein consists of the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31.Polymers

[0216] In some embodiments, an immunogenic complex described herein (e.g., a MAPS complex) comprises a polymer. In some embodiments, a component of MAPS is or comprises a polymer. The polymer may be antigenic or non- antigenic. The polymer can be made of a wide variety on substances. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the polymer is a naturally-occurring polymer. In some embodiments, the polymer is a polysaccharide. In some embodiments, the polysaccharide is an antigenic polysaccharide (e.g., an antigenic polysaccharide described herein). In some embodiments, the polysaccharide is or is derived from bacterial cells, eukaryotic cells (e.g., fungi, insect, or plant cells). In some embodiments, the polymer is or is derived from mammalian cells (e.g., virus-infected cells or cancer cells). In general, such polymers are well known in the art and are encompassed for use in the methods and compositions as disclosed herein.

[0217] In some embodiments, a polymer is a chimeric polymer comprising more than one type of polymer. For example, a polymer of an immunogenic complex as disclosed herein can comprise a first portion that is or is derived from a first polymer, and a second portion that is or is derived from a second polymer. There is no limit to the amount ofdifferent types of polymers that can be used in a single immunogenic complex. In some embodiments, a polymer is a single chain polymer. In some embodiments, a polymer is a branched polymer comprising a chain polymer and one or more branch polymers. The chain polymer can be or be derived from a first polymer. The branch polymers can be or be derived from at least 1, at least 2, at least 3 or more different polymers.

[0218] In some embodiments, a polymer is a polysaccharide. In some embodiments, the polysaccharide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 carbohydrate repeating units. In some embodiments, the polysaccharide has a molecular mass of less than 500 kDa or greater than 500 kDa. In some embodiments, the polysaccharide has a molecular mass of less than 70 kDa.

[0219] In some embodiments, a polymer is a large molecular weight polymer, e.g. , a polymer can be of an average molecular weight of between about 425 to about 500 kDa, for example, at least 300 kDa, at least 350 kDa, at least 400 kDa, at least 425 kDa, at least 450 kDa, at least 500 kDa, or greater than 500 kDa, but typically less than 500 kDa.

[0220] In some embodiments, a polymer is a small molecular weight polymer, e.g., a polymer can be of an average molecular weight of between about 60 kDa to about 90 kDa, for example, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, or greater than 100 kDa, but generally less than about 120 kDa.

[0221] In some embodiments, a polymer (e.g., a polysaccharide) is harvested and / or purified from a natural source; and in other embodiments, the polymer is synthetic. Methods to produce synthetic polymers, including synthetic polysaccharides, are known to persons of ordinary skill and are encompassed in the compositions and methods as disclosed herein.

[0222] In some embodiments, a polymer that can be used in an immunogenic complex e.g., a MAPS complex) described herein includes: dextran, polyethylene glycol- based polymers, poly(ortho ester) polymers, polyacryl carriers, PLGA, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, -amino ester polymers, polyphophoes ter (PPE), liposomes, polymerosomes, nucleic acids, polyphosphorothioated oligonucleotides, chitosan, silk, polymeric micelles, protein polymers, virus particles, virus-like particles (VLPs), or other microparticles. See, e.g., El-Sayed et al., Smart Polymer Carriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5 Exp. Op. Biol. Therapy, 23(2005). Biocompatible polymers developed for nucleic acid delivery may be adapted for use as a polymer herein. See, e.g. , Biocompatible Pol. Nucl. Acid. Deliv. (Domb el al., eds., John Wiley & Sons, Inc. Hoboken, NJ, 2011).

[0223] For example, VLPs resemble viruses, but are non-infectious because they do not contain any viral genetic materials. The expression, including recombinant expression, of viral structural proteins, such as envelope or capsid components, can result in the selfassembly of VLPs. VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV), and Flaviviridae (e.g., Hepatitis B or C viruses). VLPs can be produced in a variety of cell culture systems, including mammalian cell lines, insect cell lines, yeast, and plant cells. Recombinant VLPs are particular advantageous because the viral component can be fused to recombinant antigens as described herein.Antigenic Polysaccharides

[0224] In some embodiments, a polymer used in an immunogenic complex is or comprises an antigenic polysaccharide. In some embodiments, an antigenic polysaccharide is derived from an organism selected from the group consisting of: bacteria, archaea, viruses, or eukaryotic cells like fungi, insect, plant, or chimeras thereof. In some embodiments, an antigenic polysaccharide is a polysaccharide that is found present in or on surfaces of mammalian cells, e.g., in some embodiments cancerous cells. In some embodiments, an antigenic polysaccharide is derived from a pathogenic bacterium or virus. In some embodiments, an antigenic polysaccharide is or comprises an antigenic polysaccharide associated with a pathogen (e.g., bacteria, fungi, parasite, or virus). In some embodiments, an antigenic polysaccharide is or comprises an antigenic polysaccharide associated with a tumor. In some embodiments, an antigenic polysaccharide is or is derived from a glycoprotein. In specific embodiments, the antigenic polysaccharide is a pneumococcal capsular polysaccharide, a pneumococcal cell- wall polysaccharide, a meningococcal polysaccharide, a Haemophilus influence type b polysaccharide, a Streptococcus agalactiae polysaccharide, a Salmonella typhi Vi polysaccharide, a Klebsiella polysaccharide, a Pseudomonas polysaccharide, a Escherichia coli polysaccharide, or a Staphylococcus aureus polysaccharide. In some embodiments, an antigenic polysaccharide is derived from a tumor.

[0225] In some embodiments, an antigenic polysaccharide is, or is derived from Gram-negative bacteria and / or Gram-positive bacteria. In some embodiments, an antigenic polysaccharide is, or is derived from one or more glycoproteins. In some embodiments, one or more such glycoproteins are, or are derived from one or more viruses. In some embodiments, an antigenic polysaccharide is, or is derived from S. pneumoniae. In some embodiments, antigenic polysaccharides included in an immunogenic composition described herein are, or are derived from one or more pathogens. In some embodiments, one or more antigenic polysaccharides are, or are derived from 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) of a pathogen. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 25 serotypes or strains (variants) of a pathogen, e.g., 26, 27, 28, 29, 30, 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 serotypes or strains (variants) of a pathogen.

[0226] In some embodiments, an antigenic polysaccharide is a branched polysaccharide, or alternatively, can be a straight chain polysaccharide.

[0227] In some embodiments, an antigenic polysaccharide is a Vi antigen (Salmonella typhi capsular polysaccharide), pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharide, Hib (Haemophilus influenza type B) capsular polysaccharide, meningococcal capsular polysaccharides, the polysaccharide of Bacillus anthracis (the causative agent of anthrax), and other bacterial capsular or cell wall polysaccharides, or any combinations thereof.

[0228] In some embodiments, an antigenic polysaccharide consists of or comprises a sugar moiety. For example, in some embodiments, a polysaccharide is a Vi polysaccharide of Salmonella typhi. The Vi capsular polysaccharide has been developed against bacterial enteric infections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis. 436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993). Vi is a polymer of a-1— >-4-galacturonic acid with an N acetyl at position C-2 and variable O-acetylation at C-3. The virulence of S. typhi correlates with the expression of this molecule. Sharma et al., 101 PNAS 17492 (2004). The Vi polysaccharide vaccine of Salmonella typhi has several advantages: side effects are infrequent and mild, a single dose yields consistent immunogenicity and efficacy. Vi polysaccharide may be reliably standardized by physicochemical methods verified for other polysaccharide vaccines, Vi is stable at room temperature and it may be administeredsimultaneously with other vaccines without affecting immunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).

[0229] In some embodiments, an antigenic polysaccharide can be derived from Neisseria meningitidis, e.g. , capsular polysaccharides from at least one, two, three or four of the serogroups A, C, W, W135, or Y. In some embodiments, the polysaccharide comprises Type 5, Type 8, or any of the polysaccharides or oligosaccharides of Staphylococcus aureus.

[0230] In some embodiments, an antigenic polysaccharide can be derived from Klebsiella pneumoniae, e.g., lipopolysaccharide (LPS)-derived polysaccharides or capsular polysaccharides. In some embodiments, LPS-derived polysaccharides are O polysaccharides (OPS). In some embodiments, LPS-derived polysaccharides are core O polysaccharides (COPS). In some embodiments, the polysaccharide is from, or derived from, an OPS from Klebsiella pneumoniae serotypes 01, 02, O2ac, 03, 04, 05, 07, 08, or 012. In some embodiments, the polysaccharide is from, or derived from, a CPS from Klebsiella pneumoniae KI, K2, K10, K16, or K19.

[0231] In some embodiments, an antigenic polysaccharide is from, or derived from, Pseudomonas aeruginosa, e.g., OPS, LPS, or exopolysaccharides. In some embodiments, an antigenic polysaccharide is from, or derived from, an OPS from a Pseudomonas aeruginosa serotype selected from 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, Oi l, 012, 013, 014, 015, 016, 017, 018, 019, and 020. In some embodiments, an antigenic polysaccharide is, or is derived from, a capsular or capsular-like polysaccharide from Pseudomonas aeruginosa alginate, PsL, or Pel. In some embodiments, an antigenic polysaccharide is, or is derived from, an exopolysaccharide from Pseudomonas aeruginosa PsL.

[0232] In some embodiments, an antigenic polysaccharide present in an immunogenic complex described herein includes one or more polysaccharide from a tumor.

[0233] In some embodiments, an antigenic polysaccharide is from, or derived from, a S. pneumoniae polysaccharide. In some embodiments, an antigenic polysaccharide present in an immunogenic complex described herein includes one S. pneumoniae polysaccharide.Capsular polysaccharides are used to distinguish serotypes of S. pneumoniae. There are at least 97 distinct serotypes of .S'. pneumoniae polysaccharides, each having a different chemical structure. Figure 6 depicts exemplary structures and chemical information for certain S. pneumoniae capsular polysaccharides. All structures are from European Pharmacopoeia 9.0. Serotype designations as used herein are designations according toDanish nomenclature (Kauffmann et al, Inti. Bull. Bact. Nomenclature and Taxonomy 10:31- 41 (1960); Geno et al, Clin Microbiol Rev 28(3):871-899 (2015)).

[0234] In some embodiments, an antigenic polysaccharide present in an immunogenic complex includes one or more S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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, 11 A, 11B, 11C, HD, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 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, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

[0235] In some embodiments, an antigenic polysaccharide present in an immunogenic complex includes one or more S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, HA, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

[0236] In some embodiments, an antigenic polysaccharide present in an immunogenic complex includes one or more .S'. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 6C, 7C, 15A, 16F, 23 A, 23B, 24F, 31, 35B, and 38.Exemplary Methods of Isolating and Purifying Polysaccharides

[0237] In some embodiments, the disclosure provides methods of purifying one or more polysaccharides described herein from one or more cellular components of bacteria. In some embodiments, methods comprise purifying capsular polysaccharides from one or more cellular components of bacteria.

[0238] In some embodiments, the bacteria are Gram-negative. In some embodiments, the bacteria are Gram-positive. In some embodiments, the bacteria are 5. pneumoniae.

[0239] In some embodiments, the cellular components include protein. In some embodiments, the cellular proteins include nucleic acid. In some embodiments, the cellular components include lipids. In some embodiments, the cellular components include polysaccharides. In some embodiments, the cellular components are part of a lysate.

[0240] In some embodiments, the polysaccharide purification processes incorporate a series of ethanol precipitations, washes of crude polysaccharide preparations with ethanol, diethyl ether, and / or acetone, and drying under vacuum to furnish purified products. In some embodiments, a phenol extraction step is incorporated for polysaccharide purifications. In some embodiments, the purification process employs a CT AB (cetyltrimethyl ammonium bromide) precipitation step in addition to using ethanol and phenol precipitation steps.Exemplary Methods of Biotinylating Polysaccharides

[0241] In some embodiments, the disclosure provides methods of biotinylating one or more polysaccharides described herein. In some embodiments, the method comprises reacting purified polysaccharides with l-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of hydroxyl groups in the polysaccharides followed by the addition of amine PEG biotin under conditions that result in covalent linkage of biotin to the polysaccharides. In some embodiments, the desired level of biotinylation is achieved by varying the ratio of CDAP to polysaccharide. In some embodiments, the biotinylated polysaccharides are purified by filtration to remove process residuals such as unreacted biotin, dimethylaminopyridine, acetonitrile, cyanide and unreacted glycine. In some embodiments, the level of polysaccharide biotinylation described herein is optimized to reduce the amount of accessible biotin following MAPS complexation.Immunogenic Conjugates

[0242] Technologies and methods described herein (e.g., use of aluminum-based adjuvants at reduced concentrations) can also be applied to conjugated vaccines. In some embodiments, a conjugated vaccine is or comprises an immunogenic conjugate. As described herein, immunogenic conjugates include (i) one or more polypeptides (e.g., antigenic polypeptides) described herein conjugated to (ii) one or more polymers (e.g., antigenic polysaccharides) described herein. In some embodiments, the one or more polypeptides (e.g., antigenic polypeptides) described herein are covalently associated with the one or more polysaccharides described herein. In some embodiments, one or more polypeptide of an immunogenic conjugate comprise a carrier protein (e.g., ones described herein).

[0243] In some embodiments, one or more polysaccharides of an immunogenic conjugate comprise a capsular polysaccharide of 5. pneumoniae. In some embodiments, one or more polypeptides of an immunogenic conjugate comprise an antigenic polypeptide of .S’. pneumoniae . In some embodiments, an antigenic polypeptide of an immunogenic conjugate is or comprises a fusion protein. In some such embodiments, a fusion protein of an immunogenic conjugate is or comprises SPP2 fusion protein. In some such embodiments, a fusion protein of an immunogenic conjugate is or comprises CPI fusion protein. In some embodiments, an antigenic polypeptide or a fusion protein of an immunogenic conjugate is or comprises a carrier protein.

[0244] In some embodiments, an immunogenic conjugate comprises one or more S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, I OB, 10C, 10F, 11 A, 11B, 11C, HD, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 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, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48. In some embodiments, an immunogenic conjugate comprises one or more .S', pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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.

[0245] In some embodiments, an immunogenic conjugate comprises:(a) one or more carrier proteins described herein; and(b) one or more .S’, pneumoniae capsular polysaccharides from, or derived from, one or more (e.g. , at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, 34, or more) S. pneumoniae serotypes selected from 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, 11 A, 11B, 11C, HD, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 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, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, wherein the one or more carrier proteins are covalently attached to the one or more polysaccharides.

[0246] In some embodiments, an immunogenic conjugate comprises:(a) one or more carrier proteins described herein; and(b) one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, or 34) S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38, wherein the one or more carrier proteins are covalently attached to the one or more polysaccharides.

[0247] In some embodiments, an immunogenic conjugate comprises:(a) one or more SPP2 proteins; and(b) one or more .S', pneumoniae capsular polysaccharides from, or derived from, one or more (e.g. , at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31 , 32, 33, 34, or more) S. pneumoniae serotypes selected from 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, 11 A, 11B, 11C, HD, HE, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 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, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, wherein the one or more SPP2 proteins are covalently attached to the one or more polysaccharides.

[0248] In some embodiments, an immunogenic conjugate comprises:(a) one or more SPP2 proteins; and(b) one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, or 34) S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38, wherein the one or more SPP2 proteins are covalently attached to the one or more polysaccharides.

[0249] In some embodiments, an immunogenic conjugate comprises:(a) one or more CPI proteins; and(b) one or more S. pneumoniae capsular polysaccharides from, or derived from, one or more (e.g. , at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, 34, or more) S. pneumoniae serotypes selected from 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, 11 A, 11B, 11C, HD, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C,19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, wherein the one or more CPI proteins are covalently attached to the one or more polysaccharides.

[0250] In some embodiments, an immunogenic conjugate comprises:(a) one or more CPI proteins; and(b) one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, or 34) S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38, wherein the one or more CPI proteins are covalently attached to the one or more polysaccharides.

[0251] In some embodiments, an immunogenic composition comprises one or more immunogenic conjugates comprising:(a) one or more SPP2 proteins; and(b) one or more first S. pneumoniae capsular polysaccharides from, or derived from, one or more S. pneumoniae serotypes selected from 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, HA, 11B, 11C, HD, HE, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, wherein the one or more SPP2 proteins are covalently attached to the one or more first polysaccharides; or(c) one or more CPI proteins; and(d) one or more second S. pneumoniae capsular polysaccharides from, or derived from, one or more 5. pneumoniae serotypes selected from 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, 1 IB, 11C, 1 ID, 1 IE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, wherein the one or more CPI proteins are covalently attached to the one or more second polysaccharides.Manufacture of Immunogenic Complexes and Immunogenic Conjugates

[0252] The present disclosure includes methods for manufacturing immunogenic complexes described herein. In some embodiments, a method of manufacturing an immunogenic complex comprises complexing at least one biotinylated polymer (e.g., a biotinylated polysaccharide (e.g., a biotinylated polysaccharide described herein)) with at least one biotin-binding fusion protein described herein. In some embodiments, the present disclosure includes methods for manufacturing immunogenic conjugates described herein. In some embodiments, a method of manufacturing an immunogenic conjugate comprises conjugating at least one polymer (e.g. , a polysaccharide (e.g. , an antigenic polysaccharide)) described herein with at least one polypeptide (e.g., an antigenic polypeptide) described herein.

[0253] In some embodiments, the average (e.g., the mean) protein (e.g., antigenic protein) to polymer (e.g., polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 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 (weight / weight [w / w]). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 1:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 2:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 3:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 4:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 5:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 6:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 7: 1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 8:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality ofimmunogenic complexes or immunogenic conjugates is approximately 9:1 (w / w). In some embodiments, the average protein to polymer (e.g. , polysaccharide) ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 10:1 (w / w). Immunogenic compositions and vaccines of the invention may comprise mixtures of immunogenic complexes or immunogenic conjugates with different average protein to polymer (e.g., polysaccharide) ratios.

[0254] In some embodiments, an immunogenic composition (e.g., a vaccine) comprises a plurality of immunogenic complexes comprising a fusion protein described herein and a capsular polysaccharide. In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 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 (weight / weight [w / w]). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 1 : 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 2: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 3: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 5: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 6: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 8: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 9: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 10:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is chosen to enhance the polysaccharide immunogenicity potential (carrier or presentation function) and / or to elicit protection against, or to inhibit, a pathogen or a tumor through a protein-specific immune response.Immunogenic compositions (e.g., vaccines) of the present disclosure may comprise mixtures of immunogenic complexes with different average protein to polysaccharide ratios.

[0255] In some embodiments, a method of manufacturing immunogenic complexes comprises complexing at least one biotinylated polysaccharide with at least one biotinbinding fusion protein. In some embodiments, the fusion protein 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 to SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein 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 to SEQ ID NO: 30 or SEQ ID NO: 31.

[0256] In some embodiments, an immunogenic composition (e.g., a vaccine) comprises a plurality of immunogenic complexes comprising a fusion protein and a capsular polysaccharide. In some embodiments, the fusion protein is a SPP2 protein. In some embodiments, the fusion protein is a CPI protein. In some embodiments, the capsular polysaccharide is from or derived from S. pneumoniae having a serotype selected from 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. In some embodiments, the capsular polysaccharide is from or derived from .S', pneumoniae having a serotype selected a group that includes 30, 31 , 32, or 33 serotypes selected from 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38. In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 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 (weight / weight [w / w]). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 1: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 2:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 3:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 4: 1 (w / w). In some embodiments, the average ratio of fusion protein tocapsular polysaccharide in the plurality of immunogenic complexes is approximately 5: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 6:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 8: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 9: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 10: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is chosen to enhance the polysaccharide immunogenicity potential (carrier function) and / or to elicit protection against, or to inhibit, pneumococcal colonization by any pneumococcus (independent of polysaccharide serotype) through a protein-specific immune response. Immunogenic compositions (e.g., vaccines) of the present disclosure may comprise mixtures of immunogenic complexes with different average protein to polysaccharide ratios.

[0257] In some embodiments, an immunogenic composition (e.g., a vaccine) comprises a plurality of immunogenic complexes comprising a fusion protein and a capsular polysaccharide. In some embodiments, the fusion protein is a CPI protein. In some embodiments, the capsular polysaccharide is from or derived from S. pneumoniae having a serotype selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20B, 22F, 23F, and 33F. In some embodiments, the capsular polysaccharide is from or derived from 5. pneumoniae having a serotype selected a group that includes 20, 21, 22, or 23 serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20B, 22F, 23F, and 33F. In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 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 (weight / weight [w / w]). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 1 : 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 2: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharidein the plurality of immunogenic complexes is approximately 3: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 5: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 6: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 8:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 9: 1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 10:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is chosen to enhance the polysaccharide immunogenicity potential (carrier function) and / or to elicit protection against, or to inhibit, pneumococcal colonization by any pneumococcus (independent of polysaccharide serotype) through a protein-specific immune response. Immunogenic compositions (e.g. , vaccines) of the present disclosure may comprise mixtures of immunogenic complexes with different average protein to polysaccharide ratios.Immunogenic Compositions

[0258] Another aspect of the disclosure provides compositions that include one or more immunogenic complexes described herein. For example, an immunogenic composition, e.g. , vaccine composition, can include one or more immunogenic complexes described herein. In some embodiments, such compositions can include a plurality of one type of immunogenic complex described herein. For example, a composition can include a population of one type of immunogenic complex, where all of the immunogenic complexes include the same polypeptide (e.g. , antigenic polypeptide) and the same polymer (e.g., antigenic polysaccharide). Additionally or alternatively, such compositions can include a plurality of more than one type of immunogenic complex described herein. For example, a composition can include populations of different types of immunogenic complexes. In someembodiments, a composition can include a population of a first type of immunogenic complex and a population of a second type of immunogenic complex, where the first type and the second type of the immunogenic complex have different polypeptide (e.g. , antigenic polypeptides) and / or different polymers (e.g., antigenic polysaccharides). In some embodiments, a composition can include a population of a first type of immunogenic complex and a population of a second type of immunogenic complex, where the first type and the second type of the immunogenic complex include the same polypeptide (e.g. , antigenic polypeptide) and different polymers (e.g., antigenic polysaccharides (e.g., polysaccharides of different serotypes)).

[0259] In some embodiments, an immunogenic composition is a monovalent immunogenic composition. In some embodiments, an immunogenic composition is a polyvalent or multivalent immunogenic composition. In some embodiments, an immunogenic composition is a monovariant immunogenic composition, comprising one or more antigens from one strain or variant of a pathogen. In some embodiments, an immunogenic composition is a multivariant immunogenic composition, comprising one or more antigens from more than one strain or variant of a pathogen. In some embodiments, the valency of an immunogenic composition refers to the number of species of immunogenic complexes present in the immunogenic composition. The valency of an immunogenic described herein is not limiting with respect to the total antigens present in said immunogenic composition, or to the number of pathogen strains for which administration of said immunogenic composition may induce an immune-protective response. In a non-limiting example, a 34-valent immunogenic composition may comprise more than 34 antigenic components (e.g., peptide and / or polysaccharide components) and may induce an immunoprotective response against more than 34 pathogens, or pathogenic serotypes or strains.

[0260] In some embodiments, an immunogenic composition comprises between 1-60 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-50 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-45 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-40 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-35 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-34 species of immunogenic complexes. In someembodiments, an immunogenic composition comprises between 1-33 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-32, 1-31, or 1-30 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-30 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-30 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-24 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1 -15 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-9 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises between 1-5 species of immunogenic complexes. In some embodiments, an immunogenic composition is a polyvalent composition. In some embodiments, an 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 species of immunogenic complexes. In some embodiments, an 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 species of immunogenic complexes. In some embodiments, an immunogenic composition comprises at least 30 (including, e.g. , at least 31, at least 32, at least 33, at least 34) species of immunogenic complexes described herein. In some embodiments, an immunogenic composition comprises 30-40 species of immunogenic complexes described herein. In some embodiments, an immunogenic composition comprises 30-35 species of immunogenic complexes described herein. In some embodiments, an immunogenic composition comprises 25-50 or 25-40 species of immunogenic complexes described herein. In some embodiments, an immunogenic composition comprises no more than 60 species of immunogenix complexes described herein. In some embodiments, an immunogenic composition comprises no more than 50 species of immunogenix complexes described herein. In some embodiments, an immunogenic composition comprises no more than 45 species of immunogenix complexes described herein. In some embodiments, an immunogenic composition comprises no more than 40 species of immunogenix complexes described herein. In some embodiments, an immunogenic composition comprises no more than 35 species of immunogenix complexes described herein.

[0261] In some embodiments, an 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 species of immunogenic complexes described herein.

[0262] In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes in amounts such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition from each immunogenic complex is about the same, e.g., present in a w / w ratio of about 1:1. In some embodiments, the weight of polymers e.g., polysaccharides) in the immunogenic composition contributed by each immunogenic complex is about 0.20 pg, about 0.25 pg, about 0.5 pg, about 1 pg, about 1.5 pg, about 2 pg, about 2.5 pg, about 3 pg, about 3.5 pg, about 4 pg, about 4.5 pg, about 5 pg, about 5.5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, or about 12 pg. In some embodiments, the weight of polymers e.g., polysaccharides) in the immunogenic composition contributed by each immunogenic complex is more than 12 pg, e.g., 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, or more.

[0263] In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes in amounts such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition contributed by each immunogenic complex is different, e.g., present in a w / w ratio that is not about 1:1. In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes in amounts such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition contributed by a first immunogenic complex and a second immunogenic complex 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, such that the weight of polymers (e.g., polysaccharides) in an immunogenic composition contributed by an immunogenic complex ranges from about 0.20 pg to about 6 pg. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes, such that the weight of polymers (e.g., polysaccharides) in an immunogenic composition contributed by an immunogenic complex ranges from about 0.20 pg to about 12 pg. In some embodiments, the immunogenic composition comprises a mixture of immunogenic complexes, such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition contributed by each immunogenic complex ranges from about 0.20 pg to about 20 pg. In some embodiments, the immunogenic composition comprises a mixture of immunogeniccomplexes, such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition contributed by each immunogenic complex ranges from about 0.20 pg to about 40 pg.

[0264] In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes in amounts such that the combined weight of polymers (e.g., polysaccharides) and polypeptides in the immunogenic composition contributed by each immunogenic complex is about the same, e.g., present in a w / w proteimPS ratio of about 1: 1. In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes in amounts such that the combined weight of polymers (e.g. , polysaccharides) and polypeptides in the immunogenic composition contributed by each immunogenic complex is present in a w / w proteimPS 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.

[0265] In some embodiments, the combined weight of polymers (e.g., polysaccharides) and polypeptides in an immunogenic composition contributed by each immunogenic complex is about 0.20 pg, about 0.40 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 14 pg, about 16 pg, about 18 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, or about 110 pg.

[0266] In some embodiments, an immunogenic composition comprises two or more species of immunogenic complexes (e.g., in immunogenic compositions) in amounts such that the combined weight of polymers (e.g., polysaccharides) and polypeptides in the immunogenic composition contributed by each immunogenic complex is different, e.g., present in a w / w proteimPS ratio that is not about 1:1, e.g. , a proteimPS ratio that 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, such that the combined weight of polymers (e.g., polysaccharides) and polypeptides in the immunogenic composition contributed by each immunogenic complex ranges from about 0.4 pg to about 110 pg.

[0267] In some embodiments, an immunogenic composition described herein comprises an adjuvant.

[0268] In some embodiments, an immunogenic composition described herein is a vaccine composition.

[0269] In some embodiments, an immunogenic composition is an immunogenic composition described in WO 2020 / 056202, the contents of which are hereby incorporated by reference in their entirety for purposes described herein. In some embodiments, an immunogenic composition is an immunogenic composition described in International application PCT / US2022 / 043156, the contents of which are hereby incorporated by reference in their entirety for purposes described herein.

[0270] In some embodiments, an immunogenic composition comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more) species of immunogenic complexes selected from:(1) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 1 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(2) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 2 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(3) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 3 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(4) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 4 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(5) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 5 capsular polysaccharide, and one or more CPI SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(6) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(7) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6B capsular polysaccharide, and one or more or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(8) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 6C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(9) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6D capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(10) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6E capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(11) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 6F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(12) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 6G capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(13) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6H capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(14) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 7A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(15) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 7B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(16) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 7C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(17) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 7F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(18) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 8 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(19) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 9A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(20) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 9L capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(21) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 9N capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(22) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9V capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(23) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(24) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 10B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(25) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 10C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(26) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 10F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(27) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 11A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(28) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 1 IB capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(29) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 11C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(30) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 11D capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(31) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 1 IE capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(32) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype HF capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(33) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 12A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(34) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 12B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(35) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 12F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(36) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 13 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(37) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(38) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 15A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(39) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 15B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(40) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 15C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(41) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 15F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(42) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 16 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(43) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 16F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(44) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 17A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(45) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 17F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(46) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 18A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(47) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 18B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(48) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 18C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(49) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 18F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(50) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(51) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 19B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(52) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(53) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 19F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(54) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 20A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(55) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 20B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(56) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 21 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(57) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 22A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(58) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(59) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 23A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(60) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 23B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(61) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(62) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 24A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(63) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 24B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(64) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 24F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(65) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 25 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(66) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 25F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(67) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 27 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(68) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 28A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(69) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 28F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(70) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 29 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(71) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 31 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(72) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 32 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(73) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 32F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(74) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 33A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(75) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 33B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(76) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 33C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(77) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 33D capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(78) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 33E capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(79) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(80) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 34 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(81) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 35 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(82) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 35B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(83) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 35C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(84) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 35F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(85) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 36 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(86) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 37 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(87) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 38 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(88) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 39 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(89) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 40 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(90) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 41 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(91) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 4 IF capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(92) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 42 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(93) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 43 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(94) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 44 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(95) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 45 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(96) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 46 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(97) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 47 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(98) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 47F capsular polysaccharide, and one or more CPI or SPP2 usion proteins non-covalently complexed to the biotinylated polysaccharide; and(99) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 48 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0271] In some embodiments, an immunogenic composition comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) species of immunogenic complexes selected from:(1) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 1 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(2) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 2 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(3) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 3 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(4) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 4 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(5) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 5 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(6) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(7) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 6B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(8) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 6C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(9) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 7C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(10) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 7F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(11) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 8 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(12) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(13) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 9V capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(14) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 10A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(15) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype HA capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(16) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 12F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;Ill(17) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(18) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 15 A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(19) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 15B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(20) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 16F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(21) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(22) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 18C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(23) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(24) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(25) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 20B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(26) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(27) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 23A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(28) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 23B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(29) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(30) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 24F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(31) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 31 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(32) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 33F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(33) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 35B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide; and(34) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 38 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0272] In some embodiments, an immunogenic composition comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 21, 22, 23, or 24) species of immunogenic complexes selected from:(1) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 1 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(2) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 2 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(3) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 3 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(4) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 4 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(5) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 5 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(6) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 6A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(7) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 6B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(8) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 7F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(9) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 8 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(10) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(11) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 9V capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(12) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(13) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 11A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(14) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 12F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(15) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(16) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 15B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(17) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 17F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(18) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 18C capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(19) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19A capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(20) an immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(21) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 20B capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(22) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;(23) an immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide; and(24) an immunogenic complex comprising a biotinylated S. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI or SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0273] In some embodiments, an immunogenic composition comprises:(1) a first immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 1 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(2) a second immunogenic complex comprising a biotinylated S. pneumoniae serotype 2 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(3) a third immunogenic complex comprising a biotinylated S. pneumoniae serotype 3 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(4) a fourth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 4 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(5) a fifth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 5 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(6) a sixth immunogenic complex comprising a biotinylated S. pneumoniae serotype 6A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(7) a seventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 6B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;(8) an eighth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 6C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the...

Claims

ClaimsWe claim:

1. A pharmaceutical composition comprising an immunogenic composition comprising one or more species of immunogenic complexes, wherein the immunogenic complex of at least one of the species comprises:(a) a biotinylated polysaccharide antigen; and(b) a fusion protein comprising:(i) a biotin-binding moiety; and(ii) at least one polypeptide antigen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; and optionally wherein the pharmaceutical composition further comprises an adjuvant that is or comprises an aluminum-based adjuvant, at a reduced concentration of aluminum in the pharmaceutical composition.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises an adjuvant that is or comprises an aluminum-based adjuvant, at a reduced concentration of aluminum in the pharmaceutical composition.

3. A method of making a pharmaceutical composition, the method comprising a step of combining an immunogenic composition with an adjuvant, wherein the immunogenic composition comprises one or more species of immunogenic complexes, wherein the immunogenic complex of at least one of the species comprises:(a) a biotinylated polysaccharide antigen; and(b) a fusion protein comprising:(i) a biotin-binding moiety; and(ii) at least one antigenic polypeptide; and wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; and wherein the adjuvant is or comprises an aluminum-based adjuvant, at a reduced concentration of aluminum in the pharmaceutical composition.

4. The pharmaceutical composition or method of any one of claims 1-3, wherein the concentration of aluminum in the pharmaceutical composition is reduced relative to a reference composition.

5. The pharmaceutical composition or method of any one of claims 1-4, wherein the pharmaceutical composition is characterized in that, upon administration to a subject, it induces an immune response to (i) at least one of the polysaccharide antigens and / or (ii) at least one of the polypeptide antigens, at a level that is higher than a corresponding reference level, wherein the reference level is a level of an immune response induced in a subject following administration of a reference composition.

6. The pharmaceutical composition or method of claim 5, wherein the immune response is or comprises an antibody and / or B cell response.

7. The pharmaceutical composition or method of claim 5 or claim 6, wherein the immune response is at a level that is at least 20% higher than the corresponding reference level.

8. The pharmaceutical composition or method of any one of claims 4-7, wherein the reference composition comprises the immunogenic composition and an adjuvant that is or comprises an aluminum-based adjuvant, wherein the concentration of aluminum in the reference composition is 1.25 mg / mL or greater.

9. The pharmaceutical composition or method of any one of claims 4-8, wherein the reference composition is formulated for administration to a human.

10. The pharmaceutical composition or method of any one of claims 1-9, wherein the pharmaceutical composition is formulated for administration to a human.

11. The pharmaceutical composition or method of any one of claims 1-10, wherein the concentration of aluminum in the pharmaceutical composition is about 0.125 mg / mL to about1.25 mg / mL.

12. The pharmaceutical composition or method of any one of claims 1-11, wherein the concentration of aluminum in the pharmaceutical composition is about 0.25 to less than 1.25, about 0.25 to about 1, about 0.3 to about 0.85, or about 0.35 to about 0.75 mg / mL.

13. The pharmaceutical composition or method of any one of claims 1-12, wherein the concentration of aluminum in the pharmaceutical composition is about 0.35 mg / mL to about 0.65 mg / mL.

14. The pharmaceutical composition or method of any one of claims 1-13, wherein the concentration of aluminum in the pharmaceutical composition is about 0.5 mg / mL.

15. The pharmaceutical composition or method of any one of claims 1-14, wherein the concentration of aluminum in the pharmaceutical composition is at least 0.35 mg / mL.

16. The pharmaceutical composition or method of any one of claims 1-15, wherein the concentration of aluminum in the pharmaceutical composition is at least 0.25 mg / mL.

17. The pharmaceutical composition or method of any one of claims 1-16, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is less than 3.68: 1.

18. The pharmaceutical composition or method of any one of claims 1-17, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 3.68:1 to about 1:1, about 2.5:1 to about 1.1: 1, about2.25 to about 1.2: 1, about 2: 1 to about 1.3: 1, about 1.75: 1 to about 1.25: 1, or about 1.6: 1 to about 1.4:1.

19. The pharmaceutical composition or method of any one of claims 1-18, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 1.91: 1 to about 1.03:

120. The pharmaceutical composition or method of any one of claims 1-19, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is about 1.47: 1.

21. The pharmaceutical composition or method of any one of claims 1-20, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is at least 1.03: 1.

22. The pharmaceutical composition or method of any one of claims 1-21, wherein the ratio of the mass of aluminum to the total mass of the biotinylated polysaccharide antigens in the pharmaceutical composition is at least 0.73: 1.

23. The pharmaceutical composition or method of any one of claims 1-22, wherein the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is less than 1.22:1.

24. The pharmaceutical composition or method of any one of claims 1-23, wherein the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is about 0.64:1 to about 0.34:1.

25. The pharmaceutical composition or method of any one of claims 1-24, wherein the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is about 0.49:1.

26. The pharmaceutical composition or method of any one of claims 1-25, wherein the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is at least 0.34:1.

27. The pharmaceutical composition or method of any one of claims 1-26, wherein the ratio of the mass of aluminum to the total mass of the fusion proteins in the pharmaceutical composition is at least 0.24:1.

28. The pharmaceutical composition or method of any one of claims 1-27, wherein the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is less than 0.92:1.

29. The pharmaceutical composition or method of any one of claims 1-28, wherein the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is about 0.48:1 to about 0.26: 1.

30. The pharmaceutical composition or method of any one of claims 1-29, wherein the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is about 0.37:1.

31. The pharmaceutical composition or method of any one of claims 1-30, wherein the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is at least 0.26: 1.

32. The pharmaceutical composition or method of any one of claims 1-31, wherein the ratio of the mass of aluminum to the total mass of the immunogenic complexes in the pharmaceutical composition is at least 0.18: 1.

33. The pharmaceutical composition or method of any one of claims 1-32, wherein the aluminum-based adjuvant comprises aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate (alum), or a combination thereof.

34. The pharmaceutical composition or method of claim 33, wherein the aluminum-based adjuvant is aluminum phosphate.

35. The pharmaceutical composition or method of claim 34, wherein the concentration of the aluminum phosphate in the pharmaceutical composition is less than 5.65 mg / mL.

36. The pharmaceutical composition or method of claim 34 or claim 35, wherein the concentration of the aluminum phosphate in the pharmaceutical composition is about 2.94 mg / mL to about 1.58 mg / mL.

37. The pharmaceutical composition or method of any one of claims 34-36, wherein the concentration of aluminum phosphate in the pharmaceutical composition is about 2.26 mg / mL.

38. The pharmaceutical composition or method of any one of claims 34-37, wherein the concentration of the aluminum phosphate in the pharmaceutical composition is at least 1.58 mg / mL.

39. The pharmaceutical composition or method of any one of claims 34-38, wherein the concentration of the aluminum phosphate in the pharmaceutical composition is at least 1.13 mg / mL.

40. The pharmaceutical composition or method of any one of claims 1-39, wherein the biotinylated polysaccharide antigen comprises a bacterial polysaccharide antigen, a fungal polysaccharide antigen, a parasitic polysaccharide antigen, a viral polysaccharide antigen, a mammalian polysaccharide antigen (e.g., a tumor polysaccharide antigen).

41. The pharmaceutical composition or method of claim 40, wherein the polysaccharide antigen is or comprises a polysaccharide selected from the group consisting of: Salmonella polysaccharide, pneumococcal polysaccharides, Haemophili polysaccharides, meningococcal polysaccharides, staphylococcal polysaccharides, Bacillus anthracis polysaccharide, Streptococcus polysaccharide, Pseudomonas polysaccharide, Klebsiella polysaccharide, Cryptococcus polysaccharide, other bacterial capsular or cell wall polysaccharides, viral polysaccharides (e.g., viral glycoproteins), or combinations thereof.

42. The pharmaceutical composition or method of any one of claims 1-41, wherein the biotinylated polysaccharide antigen comprises one or more polysaccharides from Streptococcus pneumoniae.

43. The pharmaceutical composition or method of any one of claims 1-42, wherein the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9E, 9N, 9V, 10A, 10B, IOC, 10F, 11A, 11B, 11C, 11D, HE, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28 A, 28F, 29, 31 , 32A, 32F, 33 A, 33B, 33C, 33D, 33E, 33F, 34, 3 A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

44. The pharmaceutical composition or method of any one of claims 1-43, wherein the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, HA, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

45. The pharmaceutical composition or method of any one of claims 1-44, wherein the biotinylated polysaccharide antigen comprises one or more polysaccharides of Streptococcus pneumoniae each having a serotype independently selected from 6C, 7C, 15A, 16F, 23A, 23B, 24F, 31, 35B, and 38.

46. The pharmaceutical composition or method of any one of claims 1-45, wherein at least one of the polypeptide antigens is or comprises an antigen selected from the group consisting of: bacterial polypeptide antigens, fungal polypeptide antigens, parasitic polypeptide antigens, viral polypeptide antigens, and mammalian polypeptide antigens (e.g., tumor antigens).

47. The pharmaceutical composition or method of any one of claims 1-46, wherein at least one of the polypeptide antigens is or comprises an antigen selected from the group consisting of: streptococcal antigens (e.g., .S'. pneumoniae, group A, group B, and viridans antigens), tuberculosis antigens, tetanus antigens, anthrax antigens, pertussis antigens, staphylococcal antigens (e.g., .S'. aureus), Haemophilus antigens, Enterobacter, antigens, Acinetobacter antigens, Citrobacter antigens, Serratia antigens, Clostridia antigens,Campylobacter antigens, Vibriocholera antigens, Pseudomonas antigens, meningococcal antigens, Neisseria gonorrhoeae antigens, Chlamydia trachomatis antigens, Klebsiella antigens, Shigella antigens, Salmonella antigens, E. coli antigens, malaria antigens, HIV antigens, HPV antigens, influenza (e.g., seasonal or epidemic) antigens, coronavirus antigens (e.g., SARS-CoV-2 antigens), herpes (e.g., HSV) antigens, tumor antigens, and combinations thereof.

48. The pharmaceutical composition or method of any one of claims 1 -47, wherein at least one of the polypeptide antigens is or comprises a pneumococcal polypeptide antigen.

49. The pharmaceutical composition or method of any one of claims 1-48, wherein at least one of the polypeptide antigens is or comprises a pneumolysin polypeptide antigen, a SP1500 polypeptide antigen, a SP0785 polypeptide antigen, a SP0435 polypeptide antigen, or a combination thereof.

50. The pharmaceutical composition or method of any one of claims 1-49, wherein at least one species of the immunogenic complexes comprises a fusion protein comprising:(i) a biotin-binding moiety;(ii) a pneumolysin polypeptide antigen or antigenic fragment thereof; and(iii) an SP0435 polypeptide antigen or antigenic fragment thereof.

51. The pharmaceutical composition or method of claim 50, wherein the fusion protein is or comprises an amino acid sequence 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 100% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

52. The pharmaceutical composition or method of any one of claims 1-51, wherein at least one species of the immunogenic complexes comprises a fusion protein comprising:(i) a biotin-binding moiety;(ii) an SP1500 polypeptide antigen or antigenic fragment thereof; and(iii) an SP0785 polypeptide antigen or antigenic fragment thereof.

53. The pharmaceutical composition or method of claim 52, wherein the fusion protein is or comprises an amino acid sequence 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 100% identical to SEQ ID NO: 30 or SEQ ID NO: 31.

54. The pharmaceutical composition or method of any one of claims 1-53, wherein 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 species of immunogenic complexes.

55. The pharmaceutical composition or method of claim 54, wherein the immunogenic composition comprises at least 30, at least 31, at least 32, at least 33, or at least 34 species of immunogenic complexes.

56. The pharmaceutical composition or method of any one of claims 1-55, wherein the one or more species of immunogenic complexes comprise: a first plurality of species of immunogenic complexes comprising: biotinylated polysaccharide antigens selected from each of Streptococcus pneumoniae serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F, wherein each biotinylated polysaccharide antigen is non-covalently complexed with a biotin-binding moiety of a first fusion protein: wherein the first fusion protein comprises:(a) a biotin-binding moiety;(b) a first polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 7 or an antigenic fragment thereof; and(c) a second polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 9 or an antigenic fragment thereof; anda second plurality of species of immunogenic complexes comprising: biotinylated polysaccharide antigens selected from each of Streptococcus pneumoniae serotypes 2, 3, 5, 6C, 7C, 7F, 8, 9N, 10A, HA, 12F, 14, 15A, 16F, 17F, 19F, 20B, 22F, 23A, 23B, 24F, 31, 35B, and 38, wherein each biotinylated polysaccharide antigen is non-covalently complexed with a biotin-binding moiety of a second fusion protein: wherein the second fusion protein comprises:(a) a biotin-binding moiety;(b) a first polypeptide antigen comprising an amino acid sequence at least90%, 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 100% identical to SEQ ID NO: 13 or an antigenic fragment thereof; and(c) a second polypeptide antigen comprising an amino acid sequence 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 100% identical to SEQ ID NO: 11 or an antigenic fragment thereof.;57. The pharmaceutical composition or method of any one of claims 1-56, wherein the biotin-binding moiety is or comprises a rhizavidin polypeptide.

58. The pharmaceutical composition or method of any one of claims 1-57, wherein the rhizavidin polypeptide is:(i) a polypeptide comprising an amino acid sequence 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 100% identical to SEQ ID NO: 1 or a biotin-binding fragment thereof; or(ii) a polypeptide comprising an amino acid sequence 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 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3, or a biotin-binding fragment thereof.

59. The pharmaceutical composition or method of any one of claims 1-58, wherein the pharmaceutical composition is or comprises a vaccine.

60. The pharmaceutical composition or method of any one of claims 1-59, wherein the pharmaceutical composition further comprises one or more of:(a) a buffer, optionally wherein the buffer is or comprises: a histidine buffer; a succinate buffer, and / or a phosphate buffer;(b) a salt, optionally wherein the salt is or comprises sodium chloride;(c) a surfactant, optionally wherein the surfactant is or comprises polysorbate 80; and(d) a stabilizer, optionally wherein the stabilizer is or comprises sucrose.

61. The pharmaceutical composition or method of any one of claims 1-60, wherein the pharmaceutical composition further comprises a buffer that is or comprises a succinate buffer.

62. A dose of the pharmaceutical composition of any one of claims 1, 2, and 4-61.

63. The dose of claim 62, wherein the amount of aluminum in the dose is less than 0.625 mg.

64. The dose of claim 62 or claim 63, wherein the amount of aluminum in the dose is between about 0.325 mg and about 0.175 mg.

65. The dose of any one of claims 62-64, wherein the amount of aluminum in the dose is about 0.25 mg.

66. The dose of any one of claims 62-65, wherein the amount of aluminum in the dose is at least 0.175 mg.

67. The dose of any one of claims 62-66, wherein the amount of aluminum in the dose is at least 0.125 mg.

68. The dose of any one of claims 62-67, wherein the volume of the dose is about 0.5 mL.

69. A syringe comprising a dose of the pharmaceutical composition of any one of claims 1, 2, 4-61 or the dose of any one of claims 62-68.

70. A vial comprising the pharmaceutical composition of any one of claims 1, 2, 4-61 or one or more of the dose(s) of any one of claims 62-68.

71. A method comprising a step of: administering to the subject an immunologically effective amount of the pharmaceutical composition of any one of claims 1, 2, 4-61 or the dose of any one of claims 62-68.

72. A method of immunizing a subject, the method comprising a step of: administering to the subject an immunologically effective amount of the pharmaceutical composition of any one of claims 1, 2, 4-61 or the dose of any one of claims 62-68.

73. The method of claim 71 or claims 72, wherein upon administration, the pharmaceutical composition or the dose induces an immune response to (i) at least one of the polysaccharide antigens and / or (ii) at least one of the polypeptide antigens.

74. The method of claim 73, wherein the immune response is or comprises an antibody and / or B cell response.

75. The method of claim 73 or claim 74, wherein the immune response is at a level that is higher than a corresponding reference level, wherein the reference level is a level of an immune response induced in a subject following administration of a reference composition.

76. The method of claim 75, wherein the immune response is at a level that is at least 20% higher than the corresponding reference level.

77. The method of claim 75 or claim 76, wherein the reference composition comprises the immunogenic composition and an adjuvant that is or comprises an aluminum-based adjuvant, wherein the concentration of aluminum in the reference composition is 1.25 mg / mL or greater.

78. The method of any one of claims 71-77, wherein the subject is a human subject.

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