EP4680273A1

EP4680273A1 - Immunogenic compositions

Info

Publication number: EP4680273A1
Application number: EP24771805.9A
Authority: EP
Inventors: Bounthon Loun; Taylor C. STEVENSON
Original assignee: Affinivax Inc
Current assignee: Affinivax Inc
Priority date: 2023-03-16
Filing date: 2024-03-15
Publication date: 2026-01-21
Also published as: CN120826235A; JP2026509511A; WO2024192346A1

Abstract

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

Description

IMMUNOGENIC COMPOSITIONS

Cross 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 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).

[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. In some 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 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 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 the pharmaceutical 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 immunogenic complexes 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 no more 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%, 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.

[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. In particular 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 is formed 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 Types serotypes, 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 additional polysaccharide 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 through action 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, electrostatic interactions, 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, such quantity 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” refers to 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, an immunoprotective 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, a substance 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” is intended 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 simultaneously with 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 of different 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 least 3, 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 reference polypeptide 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 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).

[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 be desirable 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-covalently complexed 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 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 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 antigenic polypeptides 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 some embodiments, 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 pathogen other 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 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: 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 other DNA 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 to any 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 the sequence 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. pneumoniae polypeptide 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 acid residues. 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 comprises a 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 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: 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, or twenty 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 forming hydrogen 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 the above 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 H_n, 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 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.

[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 H_n, 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 of

MGWSCIILFLVATATGVHS (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"¹⁵ M) 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-binding moiety 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 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 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 SPP2 fusion 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%, 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, 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 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: 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%, 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 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 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: 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 of different 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 administered simultaneously 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 to Danish 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 of immunogenic 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 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.

[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 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 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 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 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 some embodiments, 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 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 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;

(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;

(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 biotinylated polysaccharide;

(9) a ninth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 7C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(10) a tenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 7F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(11) an eleventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 8 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(12) a twelfth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(13) a thirteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9V capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(14) a fourteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(15) a fifteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 11A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(16) a sixteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 12F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(17) a seventeenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 14 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (18) an eighteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 15A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(19) a nineteenth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 15B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(20) a twentieth immunogenic complex comprising a biotinylated S. pneumoniae serotype 16F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(21) a twenty-first immunogenic complex comprising a biotinylated S. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(22) a twenty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 18C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(23) a twenty-third immunogenic complex comprising a biotinylated S. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(24) a twenty-fourth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(25) a twenty-fifth immunogenic complex comprising a biotinylated S. pneumoniae serotype 20B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(26) a twenty-sixth immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(27) a twenty- seventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 23 A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (28) a twenty-eighth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 23B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(29) a twenty-ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(30) a thirtieth immunogenic complex comprising a biotinylated S. pneumoniae serotype 24F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(31) a thirty-first immunogenic complex comprising a biotinylated S. pneumoniae serotype 31 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(32) a thirty-second immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(33) a thirty-third immunogenic complex comprising a biotinylated S. pneumoniae serotype 35B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; and

(34) a thirty-fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 38 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0274] In some embodiments, an immunogenic composition comprises:

(1) a first immunogenic complex comprising a biotinylated S. pneumoniae serotype 6C capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(2) a second immunogenic complex comprising a biotinylated S. pneumoniae serotype 7C capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(3) a third immunogenic complex comprising a biotinylated S. pneumoniae serotype 15 A capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide; (4) a fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 16F capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(5) a fifth immunogenic complex comprising a biotinylated S. pneumoniae serotype 23A capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(6) a sixth immunogenic complex comprising a biotinylated S. pneumoniae serotype 23B capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(7) a seventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 24F capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(8) an eighth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 31 capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(9) a ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 35B capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(10) a tenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 38 capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(11) an eleventh immunogenic complex comprising a biotinylated 5. pneumoniae serotype 1 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(12) a twelfth 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;

(13) a thirteenth 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; (14) a fourteenth 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;

(15) a fifteenth 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;

(16) a sixteenth 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;

(17) a seventeenth 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;

(18) an eighteenth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 7F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(19) a nineteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 8 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(20) a twentieth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(21) a twenty-first immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 9V capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(22) a twenty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(23) a twenty-third immunogenic complex comprising a biotinylated S. pneumoniae serotype 11A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (24) a twenty-fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 12F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(25) a twenty-fifth immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(26) a twenty-sixth immunogenic complex comprising a biotinylated S. pneumoniae serotype 15B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(27) a twenty- seventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(28) a twenty-eighth immunogenic complex comprising a biotinylated S. pneumoniae serotype 18C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(29) a twenty-ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(30) a thirtieth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 19F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(31) a thirty-first immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 20B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(32) a thirty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(33) a thirty-third immunogenic complex comprising a biotinylated .S', pneumoniae serotype 23F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; and (34) a thirty-fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0275] 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 SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(2) a second immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 4 capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(3) a third immunogenic complex comprising a biotinylated S. pneumoniae serotype 6A capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(4) a fourth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 6B capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(5) a fifth immunogenic complex comprising a biotinylated S. pneumoniae serotype 9V capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(6) a sixth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 15B capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(7) a seventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 18C capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(8) an eighth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 19A capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(9) a ninth immunogenic complex comprising a biotinylated .S’, pneumoniae serotype 23F capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide; (10) a tenth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 33F capsular polysaccharide, and one or more SPP2 fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(11) an eleventh immunogenic complex comprising a biotinylated 5. pneumoniae serotype 2 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(12) a twelfth 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;

(13) a thirteenth 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;

(14) a fourteenth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 6C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(15) a fifteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 7C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(16) a sixteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 7F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(17) a seventeenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 8 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(18) an eighteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(19) a nineteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (20) a twentieth immunogenic complex comprising a biotinylated .S', pneumoniae serotype HA capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(21) a twenty-first immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 12F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(22) a twenty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(23) a twenty-third immunogenic complex comprising a biotinylated S. pneumoniae serotype 15A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(24) a twenty-fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 16F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(25) a twenty-fifth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(26) a twenty-sixth immunogenic complex comprising a biotinylated S. pneumoniae serotype 19F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(27) a twenty- seventh immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 20B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(28) a twenty-eighth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 22F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(29) a twenty-ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 23 A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (30) a thirtieth immunogenic complex comprising a biotinylated S. pneumoniae serotype 23B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(31) a thirty-first immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 24F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(32) a thirty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 31 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(34) a thirty-fourth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 38 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0276] In some embodiments, an immunogenic composition comprises:

(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 7F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(9) a ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 8 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(10) a tenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9N capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(11) an eleventh immunogenic complex comprising a biotinylated .S', pneumoniae serotype 9V capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(12) a twelfth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 10A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(13) a thirteenth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 11A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(14) a fourteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 12F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(15) a fifteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (16) a sixteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 15B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(17) a seventeenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(18) an eighteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 18C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(19) a nineteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(20) a twentieth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 19F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(21) a twenty-first immunogenic complex comprising a biotinylated .S', pneumoniae serotype 20B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(22) a twenty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(23) a twenty-third immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; and

(24) a twenty-fourth immunogenic complex comprising a biotinylated S. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide.

[0277] 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 3 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 4 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 5 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 6A 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 6B 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 7F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(8) an eighth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 8 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(9) a ninth immunogenic complex comprising a biotinylated S. pneumoniae serotype 9N capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(10) a tenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 9V capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (11) an eleventh immunogenic complex comprising a biotinylated S. pneumoniae serotype 10A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(12) a twelfth immunogenic complex comprising a biotinylated .S'. pneumoniae serotype 1 1 A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(13) a thirteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 12F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(14) a fourteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 14 capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(15) a fifteenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 15B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(16) a sixteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 17F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(17) a seventeenth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 18C capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(18) an eighteenth immunogenic complex comprising a biotinylated 5. pneumoniae serotype 19A capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(19) a nineteenth immunogenic complex comprising a biotinylated S. pneumoniae serotype 19F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(20) a twentieth immunogenic complex comprising a biotinylated .S', pneumoniae serotype 20B capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; (21) a twenty-first immunogenic complex comprising a biotinylated S. pneumoniae serotype 22F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide;

(22) a twenty-second immunogenic complex comprising a biotinylated S. pneumoniae serotype 23F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide; and

(23) a twenty-third immunogenic complex comprising a biotinylated S. pneumoniae serotype 33F capsular polysaccharide, and one or more CPI fusion proteins non-covalently complexed to the biotinylated polysaccharide.

Pharmaceutical Compositions

[0278] In some embodiments, a composition described herein is formulated into a pharmaceutical composition. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an adjuvant.

[0279] Optimal amounts of components for a particular pharmaceutical composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial immunization, subjects can receive one or several booster immunizations adequately spaced in time.

[0280] The immunogenic complexes described herein, and/or preparations thereof, may be formulated in a unit dosage form for ease of administration and uniformity of dosage. The specific therapeutically effective dose level for any particular subject or organism may depend upon a variety of factors including the severity or degree of risk of infection; the activity of the specific pharmaceutical composition employed; other characteristics of the specific pharmaceutical composition employed; the age, body weight, general health, sex of the subject, diet of the subject, pharmacokinetic condition of the subject, the time of administration (e.g., with regard to other activities of the subject such as eating, sleeping, receiving other medicines including other pharmaceutical composition doses, etc.), route of administration, rate of excretion of the specific pharmaceutical composition employed; pharmaceutical compositions used in combination or coincidental with the pharmaceutical composition employed; and like factors well known in the medical arts. [0281] Immunogenic complexes for use in accordance with the present disclosure may be formulated into compositions (e.g. , pharmaceutical compositions) according to known techniques. In some embodiments where a pharmaceutical composition comprises a vaccine composition, vaccine preparation is generally described in Vaccine Design (Powell and Newman, 1995). For example, an immunologically effective amount of a vaccine composition can be formulated together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials.

[0282] In general, pharmaceutically acceptable carrier(s) include solvents, dispersion media, and the like, which are compatible with pharmaceutical administration. For example, materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose, dextrose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; polyols such as glycerol, propylene glycol, and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator (Martin, 1975).

[0283] Pharmaceutical compositions may be formulated by combining one or more of the immunogenic complexes disclosed herein with carriers and/or other optional components by any available means including, for example, conventional mixing, granulating, dissolving, lyophilizing, or similar processes.

[0284] Pharmaceutical compositions useful in the provided methods may be lyophilized up until they are about to be used, at which point they are extemporaneously reconstituted with diluent. In some embodiments, pharmaceutical compositions or components thereof are lyophilized in the presence of one or more other components (e.g., adjuvants), and are extemporaneously reconstituted with saline solution. Alternatively, individual components, or sets of components may be separately lyophilized and/or stored (e.g., in a vaccination kit), the components being reconstituted and either mixed prior to use or administered separately to the subject. [0285] Lyophilization can produce a more stable composition (e.g., by preventing or reducing breakdown of polysaccharide antigens). Lyophilizing of pharmaceutical compositions or components thereof is well known in the art. Typically, a liquid pharmaceutical composition or component thereof is freeze dried, often in the presence of an anti-caking agent (such as, for example, sugars such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10- 200 mg/ml. Lyophilization typically occurs over a series of steps, for instance a cycle starting at -69° C, gradually adjusting to -24°C over 3 h, then retaining this temperature for 18 h, then gradually adjusting to -16°C over 1 h, then retaining this temperature for 6 h, then gradually adjusting to +34°C over 3 h, and finally retaining this temperature over 9 h.

[0286] In some embodiments, a pharmaceutical composition is a liquid. In some embodiments, the liquid is a reconstituted lyophylate. In some embodiments, a pharmaceutical composition has a pH of about 5, about 6, about 7, or about 8. In some embodiments, a pharmaceutical composition has a pH between about 5 and about 7.5. In some embodiments, a pharmaceutical composition has a pH between 5 and 7.5. In some embodiments, a pharmaceutical composition has a pH between about 5.3 and about 6.3. In some embodiments, a pharmaceutical composition has a pH between 5.3 and 6.3. In some embodiments, a pharmaceutical composition has a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0287] Pharmaceutical compositions or components thereof for use in accordance with the present disclosure may be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or immune- stimulating complexes (ISCOMs).

[0288] In certain situations, it may be desirable to prolong the effect of a pharmaceutical composition for use in accordance with the present disclosure, for example by slowing the absorption of one or more pharmaceutical composition components. Such delay of absorption may be accomplished, for example, by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the product then depends upon its rate of dissolution, which in turn, may depend upon size and form. Alternatively, or additionally, delayed absorption may be accomplished by dissolving or suspending one or more pharmaceutical composition components in an oil vehicle. Injectable depot forms can also be employed to delay absorption. Such depot forms can be prepared by forming microcapsule matrices of one or more pharmaceutical composition components a biodegradable polymers network. Depending upon the ratio of polymer to pharmaceutical composition component, and the nature of the particular polymer(s) employed, the rate of release can be controlled.

[0289] Examples of biodegradable polymers that can be employed in accordance with the present invention include, for example, poly(orthoesters) and poly (anhydrides). One particular exemplary polymer is polylactide-polyglycolide.

[0290] Depot injectable formulations may also be prepared by entrapping the product in liposomes or microemulsions, which are compatible with body tissues.

[0291] Polymeric delivery systems can also be employed in non-depot formulations including, for example, oral formulations. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, etc., can be used in oral formulations. Polysaccharide antigens or conjugates may be formulated with such polymers, for example to prepare particles, microparticles, extrudates, solid dispersions, admixtures, or other combinations in order to facilitate preparation of useful formulations (e.g., oral).

[0292] Pharmaceutical compositions for use in accordance with the present invention include immunogenic compositions, and may additionally include one or more additional active agents (z.<?., agents that exert a biological effect - not inert ingredients). For example, it is common in pharmaceutical composition preparation to include one or more adjuvants. It will be appreciated that such additional agents may be formulated together with one or more other pharmaceutical composition components, or may be maintained separately and combined at or near the time of administration. In some embodiments, such additional components may be administered separately from some or all of the other pharmaceutical composition components, within an appropriate time window for the relevant effect to be achieved.

Adjuvants

[0293] The immunogenic compositions (e.g., vaccine compositions) and pharmaceutical compositions described herein may include an adjuvant. Adjuvants, generally, are agents that enhance the immune response to an antigen. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et al., 2003). In most immunogenic composition or pharmaceutical composition formulations, the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen. Vaccine delivery systems are often particulate formulations, e.g. , emulsions, microparticles, immune- stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and/or matrices, and liposomes. Tn contrast, immunostimulatory adjuvants are sometimes from or derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), or CpG-containing DNA, which activate cells of the innate immune system.

[0294] Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include aluminum-based adjuvants (e.g., alum salts), such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. Non-limiting examples of organic adjuvants include cholera toxin/toxoids, other enterotoxins/toxoids or labile toxins/toxoids of Gram-negative bacteria, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0295] Adjuvants may also be classified by the response they induce. In some embodiments, an adjuvant induces generation, proliferation, or activation of THI cells or TH2 cells. In other embodiments, an adjuvant induces generation, proliferation, or activation of B cells. In yet other embodiments, an adjuvant induces activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.

[0296] In certain embodiments, the adjuvant induces the generation, proliferation, or activation of TH17 cells. The adjuvant may promote the CD4+ or CD8+ T cells to secrete IL- 17. In some embodiments, an adjuvant that induces the generation, proliferation, or activation of Tn 17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL- 17 levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce TH 17 generation, proliferation, or activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the generation, proliferation, or activation of TH17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL- 17 may be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coll, or ETEC), activate a TH 17 response. Thus, in some embodiments, the adjuvant is a toxin or toxoid. Mutant derivates of labile toxin (toxoids) that are active as adjuvants but significantly less toxic can be used as well. Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al., 1995) and LTK63 (Williams et al., 2004), LT-G192 (Douce et al., 1999), and LTR72 (Giuliani et al., 1998).

[0297] In some embodiments, the adjuvant comprises a VLP (virus-like particle).

One such adjuvant platform, Alphavirus replicons, induces the activation of TH17 cells using alphavirus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL- 17 or a cytokine that stimulates IL- 17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, an immunogenic composition (e.g., vaccine) formulation is administered to a subject in combination with a nucleic acid encoding a cytokine.

[0298] Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a TH17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram-negative bacteria, such as S. minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans et al, 2003. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka et al, 2007). Various TLR agonists (including TLR-4 agonists) have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR- activating adjuvant is R848 (resiquimod).

[0299] In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponari tree. A saponin extract from a biological source can be further fractionated (e.g. , by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.

[0300] In certain embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.

[0301] Adjuvants may be covalently or non-covalently bound to antigens. In some embodiments, the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen-presenting cells (APCs). In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses e.g. , see Wu et al., 2005).

[0302] In some embodiments, immunogenic complexes described herein are formulated and/or administered in combination with an adjuvant. In some embodiments, such an adjuvant is or comprises an aluminum-based adjuvant (e.g., an aluminum salt); see e.g., Danielsson and Eriksson, “Aluminum adjuvants in vaccines - a way to modulate the immune response”, Semin Cell Dev Biol, 115:3-9 (2021) and HogenEsch et al., “Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want”, NPJ Vaccines, 3:51 (2018), the contents of which are incorporated herein by reference in their entirety for purposes described herein. Examples of an aluminum-based adjuvant include, but are not limited to, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate (alum), aluminum hydroxide phosphate, aluminum hydroxy phosphate sulfate, and combinations thereof. In some embodiments, an aluminum-based adjuvant is or comprises aluminum phosphate. In some embodiments, an aluminum -based adjuvant is or comprises aluminum phosphate.

[0303] Aluminum phosphate (including both anhydrous and hydrated forms) is often referred to for convenience as “A1PO4”, although hydrated forms (hydroxyphosphates) can be distinguished from anhydrous A1PO4 by the presence of hydroxyl groups (Al(OH), (PO4), e.g. A1(OH)(PO4)). In some embodiments, the aluminum phosphate is aluminum hydroxyphosphate (e.g., amorphous aluminum hydroxyphosphate). In some embodiments, aluminum hydroxyphosphate can contain a small amount of sulfate (aluminum hydroxyphosphate sulfate). In some embodiments, the aluminum phosphate is aluminum orthophosphate (also known as "aluminum monophosphate"). In some embodiments, the aluminum phosphate is an aluminum phosphate gel. Aluminum phosphate can be a precipitate of insoluble aluminum phosphate (amorphous, semi-crystalline, or crystalline) which may be prepared by mixing soluble aluminum salts and phosphoric acid salts, e.g., sodium phosphate or potassium phosphate. In some embodiments, the aluminum phosphate is amorphous (e.g., amorphous hydroxyphosphate). Aluminum hydroxy phosphate is not a stoichiometric compound and its hydroxyl and phosphate composition depends on precipitation reactants and conditions. In some embodiments, the Phosphate:Aluminum (P:A1) weight/weight (w/w) of an aluminum phosphate adjuvant can be between 2:1 to 4:1, suitably between 2.5:1 to 3.6:1, or between 3:1 to 3.6: 1. Aluminum content may be determined by atomic absorption spectrophotometry with nitrous flame, see for example May et al. J. Biol. Stand. 12(2): 175-83 (1984), or by inductively coupled plasma optical emission spectroscopy (ICP-EOS). Completeness of adsorption of a polypeptide (e.g., an antigenic polypeptide) onto aluminum phosphate can be measured by measuring the supernatant of centrifuged samples via Lowry and comparing the total amount of protein in the sample (measured before adsorption occurs or by desorbing adsorbed antigen) to the amount which remains in the supernatant after centrifugation; see e.g., Chapter 4 of Methods in Molecular Medicine, Vol. 42 (edited by D.T. O’Hagan) Vaccine Adjuvants Preparation Methods and Research Protocols. Particle size of a polypeptide (e.g., an antigenic polypeptide) adsorbed onto aluminum phosphate, can be measured by SLS (static light scattering); see e.g., E. Lindblad, Immunology and Cell Biology 82: 497-505 (2004). Adsorptive capacities of between 0.7- 1.5 mg protein per mg aluminum at pH 7.4 have been reported for aluminum phosphate adjuvants. The amount of adsorption for any formulation will depend on a combination of parameters including the adjuvant’s point of zero charge (PZC; the pH at which a surface has zero net charge), the salt concentration and pH during formulation, the adjuvant concentration, the concentration and the isoelectric point (pl) of the polypeptide antigen. The impact of each of these parameters on adsorption can be readily assessed.

[0304] One aspect of the present disclosure, among other things, provides an insight that the immunogenicity of pharmaceutical compositions described herein can be improved by varying the amount or concentration of an adjuvant used in a pharmaceutical composition. For example, the present disclosure, among other things, provides an insight that pharmaceutical compositions having a reduced amount or concentration of aluminum (e.g., from an aluminum-based adjuvant) may be desirable to increase immunogenicity to an antigenic polypeptide and/or a polysaccharide antigen present in the pharmaceutical composition. In some embodiments, a pharmaceutical composition described herein includes an aluminum-based adjuvant, at a reduced concentration of aluminum in the pharmaceutical composition. In some embodiments, a pharmaceutical composition described herein comprises a reduced concentration of aluminum. In some embodiments, a pharmaceutical composition described herein comprises a reduced concentration of aluminum, where the aluminum is from an aluminum-based adjuvant.

[0305] 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.

[0306] In some embodiments, a reduced concentration of aluminum in a pharmaceutical composition described is characterized relative to a reference composition. In some embodiments, a reference composition is a composition that comprises an immunogenic composition described herein and an aluminum-based adjuvant, where the concentration of aluminum in the reference composition is 1.25 mg/mL or greater (e.g, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mg/mL, or greater). In some embodiments, the reference composition is formulated for administration to a human.

[0307] In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized in that upon administration to a subject (e.g., a human), it induces an immune response against (i) at least one polysaccharide antigen and/or (ii) at least one polypeptide antigen present in the pharmaceutical composition. In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is (i) an antibody or B cell response and (ii) a T cell response. In some embodiments, the immune response is at a level that is at least 10% higher (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, or higher) than a corresponding reference level. In some embodiments, the immune response is at a level that is at least 1.1 -fold higher (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, or higher) than a corresponding reference level. In some embodiments, such a corresponding reference level is a level of an immune response elicited by a reference composition. In some embodiments, such a reference composition is a composition that comprises an immunogenic composition described herein and an aluminum-based adjuvant, where the concentration of aluminum in the reference composition is 1.25 mg/mL or greater (e.g, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mg/mL, or greater). In some embodiments, the reference composition is formulated for administration to a human. The present disclosure, among other things, also includes methods of increasing the level of an immune response induced by a pharmaceutical composition described herein by reducing the amount or concentration of an aluminum-based adjuvant relative to a reference composition.

[0308] In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum that is less than 1.25 mg/mL (e.g., less than 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, or 0.1 mg/mL). In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum that is at least 0.1 mg/mL (e.g., at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2 mg/mL, or greater). In some embodiments, the concentration of aluminum that is at least 0.25 mg/mL. In some embodiments, the concentration of aluminum that is at least 0.35 mg/mL. In some embodiments, a pharmaceutical composition described herein containing an aluminum- based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum that is about 0.1 to less than 1.25 mg/mL. In some embodiments, the concentration of aluminum that is about 0.1 to about 0.25, about 0.25 to about 0.35, about 0.35 to about 0.5, about 0.5 to about 0.75, about 0.75 to about 1, or about 1 to about 1.25 mg/mL. In some embodiments, the concentration of aluminum that is about 0.45 to about 0.55, about 0.4 to about 0.6, about 0.35 to about 0.65, about 0.3 to about 0.7, about 0.25 to about 0.75, about 0.2 to about 0.8, about 0.15 to about 0.85, about 0.1 to about 0.9 mg/mL. In some embodiments, the concentration of aluminum that is about 0.25 to about 1.25, about 0.25 to about 1, about 0.3 to about 0.85, or about 0.35 to about 0.75 mg/L. In some embodiments, the concentration of aluminum that is about 0.35 to about 0.65 mg/mL. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum that is about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65 mg/mL. In some embodiments, the concentration of aluminum is about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the concentration of aluminum is about 0.5 mg/mL. In some embodiments, the concentration of aluminum can be lower than 0.25 mg/mL. In some embodiments, the concentration of alumimum can be lower than 0. 1 mg/mL.

[0309] Unless otherwise stated, the concentration of aluminum as provided herein is characterized as the mass amount of the elemental aluminum relative to the solution volume of a pharmaceutical composition comprising an immunogenic composition described herein.

[0310] Aluminum-based adjuvants differ between each other in the percentage of their mass that comprises elemental aluminum. For example, anhydrous aluminum phosphate is approximately 22.125% elemental aluminum by mass. Accordingly, those of skill in the art will appreciate that in order to achieve a particular amount or concentration of aluminum in a preparation, different amounts or concentrations of an aluminum-based adjuvant will be needed depending on the particular aluminum-based adjuvant being used. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum phosphate that is less than 5.6 mg/mL (e.g., less than 5.6, 5.4, 5.2, 5.0, 4.8, 4.6, 4.4, 4.2, 4.0, 3.8, 3.6, 3.4, 3.2, 3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, or 0.4 mg/mL). In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum phosphate that is at least 0.4 mg/mL (e.g., at least 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4 mg/mL, or higher). In some embodiments, the concentration of aluminum phosphate is at least 1.13 mg/mL. In some embodiments, the concentration of aluminum phosphate is at least 1.58 mg/mL. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum phosphate that is about 0.4 mg/mL to about 5.6 mg/mL. In some embodiments, the concentration of aluminum phosphate is about 0.4 to about 1, about 1 to about 1.6, about 1.6 to about 2.2, about 2.2 to about 2.8, about 2.8 to about 3.4, about 3.4 to about 4, about 4 to about 4.6, about 4.6 to about 5.2, or about 5.2 to about 5.6 mg/mL. In some embodiments, the concentration of aluminum phosphate is about 2.1 to about 2.5, about 1.8 to about 2.7, about 1.6 to about 2.3, about 1.4 to about 3.2, about 1.1 to about 3.4, about 0.9 to about 3.6, about 0.7 to about 3.8, or about 0.5 to about 4.1 mg/mL. In some embodiments, the concentration of aluminum phosphate is about 2.09 to about 2.49, about 1.81 to about 2.71, about 1.58 to about 2.94, about 1.36 to about 3.16, about 1.13 to about 3.39, about 0.9 to about 3.62, about 0.68 to about 3.84, or about 0.45 to about 4.07 mg/mL. In some embodiments, the concentration of aluminum phosphate is about 1.58 to about 2.94 mg/mL. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum phosphate that is about 1.6, about 1.7, about 1.8, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, or about 2.9 mg/mL. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition has a concentration of aluminum phosphate that is about 1.58, about 1.81, about 2.03, about 2.26, about 2.49, about 2.71, about 2.94 mg/mL. In some embodiments, the concentration of aluminum phosphate is about 2.3 mg/mL. In some embodiments, the concentration of aluminum phosphate 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 is about 2.26 mg/mL.

[0311] In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) that is less than 3.68:1 (e.g., less than 3.68:1, 3.5:1, 3:1, 2.5:1, 2: 1, 1.75: 1, 1.5:1, 1.25:1, 1:1, 0.75:1, 0.5: 1, or 0.25: 1). In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) that is at least about 0.25: 1 (e.g., 0.25:1, 0.5:1, 0.75:1, 1: 1, 1.25:1, 1.5:1, 1.75: 1, 2:1, 2.25:1, 2.5: 1, 2.75: 1, 3: 1, 3.25: 1, 3.5, or higher). In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is at least 1.03:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is at least 0.73:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) that is about 0.25:1 to about 3.68:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is about 0.25: 1 to about 0.5:1, about 0.5:1 to about 1 :1, about 1:1 to about 1.5:1, about 1.5:1 to about 2:1, about 2: 1 to about 2.5:1, about 2.5: 1 to about 3:1, about 3:1 to about 3.5: 1, or about 3.5:1 to about 3.68: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is about 1.3: 1 to about 1.6: 1, about 1.2: 1 to about 1.8:1, about 1.0:1 to about 1.9:1, about 0.9: 1 to about 2.1;1, about 0.7: 1 to about 2.2:1, about 0.6:1 to about 2.4;1, about 0.4:1 to about 2.5:1, or about 0.3: 1 to about 2.7:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens is about 1.32:1 to about 1.62:1, about 1.18:1 to about 1.76: 1, about 1.03:1 to about 1.91: 1, about 0.88: 1 to about 2.06;l, about 0.74: 1 to about 2.21:1, about 0.59:1 to about 2.35; 1, about 0.44: 1 to about 2.5:1, or about 0.29: 1 to about 2.65:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) 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 polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is about 1.03: 1 to about 1.91:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) that is about 1.0:1, about 1.1:1, about 1.12:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, or about 1.9:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) that is about 1.03:1, about 1.18:1, about 1.32:1, about 1.47:1, about 1.62:1, about 1.76:1, or about 1.91:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) 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 polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is about 1.5:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polysaccharide antigens (e.g., biotinylated polysaccharide antigens) is about 1.47:1.

[0312] In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) that is less than 1.22:1 (e.g., less than 1.22:1, 1.2: 1, 1.1: 1, 1: 1, 0.9:1, 0.8: 1, 0.7: 1, 0.6: 1, 0.5:1, 0.4: 1, 0.3: 1, 0.2: 1, or 0.1: 1). In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) that is at least about 0.1:1 (e.g., 0.1:1, 0.2:1, 0.3:1, 0.4: 1, 0.5: 1, 0.6: 1, 0.7:1, 0.8: 1, 0.9:1, 1: 1, 1.1:1, 1.2: 1, or higher). In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is at least 0.34:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is at least 0.24:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) that is about 0.1:1 to about 1.22:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.1:1 to about 0.3: 1, about 0.3: 1 to about 0.5: 1, about 0.5: 1 to about 0.7:1, about 0.7: 1 to about 0.9: 1, about 0.9: 1 to about 1.1: 1, or about 1.1 :1 to about 1 .22: 1 . In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.4:1 to about 0.5: 1, about 0.4: 1 to about 0.6:1, about 0.3:1 to about 0.6:1, about 0.3: 1 to about 0.7:1, about 0.2:1 to about 0.8:1, or about 0. 1: 1 to about 0.9: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.44:1 to about 0.54: 1, about 0.39:1 to about 0.59:1, about 0.34:1 to about 0.64:1, about 0.29:1 to about 0.69:1, about 0.25:1 to about 0.74:1, about 0.2:1 to about 0.78:1, about 0.15:1 to about 0.83:1, or about 0.1:1 to about 0.88:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.34:1 to about 0.64: 1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) that is about 0.3: 1, about 0.4: 1, about 0.5: 1, about 0.6: 1, or about 0.7: 1. In some embodiments, a pharmaceutical composition described herein containing an aluminum- based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) that is about 0.34: 1, about 0.39:1, about 0.44:1, about 0.49: 1, about 0.54: 1, about 0.59: 1, or about 0.64:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) 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 polypeptides (e.g., fusion proteins) is about 0.5:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.49: 1.

[0313] In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of immunogenic complexes that is less than 0.92:1 (e.g., less than 0.92:1, 0.9: 1, 0.8:1, 0.7: 1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2: 1, or 0.1:1). In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of immunogenic complexes that is at least about 0.1: 1 (e.g., 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or higher). In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes is at least 0.26:1. In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes is at least 0.18: 1 . In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of immunogenic complexes that is about 0.1: 1 to about 0.92: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes is about 0.1:1 to about 0.2:1, about 0.2:1 to about 0.3:1, about 0.3:1 to about 0.4:1, about 0.4:1 to about 0.5:1, about 0.5:1 to about 0.6: l, about 0.6:l to about 0.7:l, about 0.7:1 to about 0.8: 1, or about 0.8:1 to about 0.92: 1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.3: 1 to about 0.4: 1, about 0.2: 1 to about 0.5:1, about 0.2:1 to about 0.6:1, or about 0.1:1 to about 0.6:1. In some embodiments, the ratio of the mass of aluminum to the total mass of polypeptides (e.g., fusion proteins) is about 0.33:1 to about 0.41:l, about 0.30: 1 to about 0.44: 1, about 0.26: l to about 0.48: 1, about 0.22: 1 to about 0.52: 1, about 0.19:1 to about 0.56:1, about 0.15:l to about 0.59: l, or about 0.i l: ! to about 0.63:1. In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes is about 0.26:1 to about 0.48:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of immunogenic complexes that is about 0.2:1, about 0.3:1, about 0.4:1, or about 0.5:1. In some embodiments, a pharmaceutical composition described herein containing an aluminum-based adjuvant with a reduced amount or concentration of aluminum in the pharmaceutical composition is characterized by a ratio of the mass of aluminum to the total mass of immunogenic complexes that is about 0.26:1, about 0.3:1, about 0.33: 1, about 0.37:1, about 0.41:1, about 0.44:1, or about 0.48:1. In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes is about 0.4:1. In some embodiments, the ratio of the mass of aluminum to the total mass of immunogenic complexes 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 immunogenic complexes is about 0.37: 1.

[0314] In some embodiments, the same adjuvant or mixture of adjuvants is present in each dose of an immunogenic composition or pharmaceutical composition. Optionally, however, an adjuvant may be administered with the first dose of an immunogenic composition or pharmaceutical composition and not with subsequent doses (i.e., booster shots). Alternatively, a strong adjuvant may be administered with the first dose of an immunogenic composition or pharmaceutical composition and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human subjects, non-human animals, or both.

[0315] Immunogenic compositions and pharmaceutical compositions for use in accordance with the present disclosure may include, or be administered concurrently with, other antimicrobial, antiviral, or anti-inflammatory therapies. For example, such immunogenic compositions and pharmaceutical compositions may include or be administered with one or more agents that kills or retards growth of a pathogen. Such agents include, for example, remdesivir, lopinavir and/or ritonavir (e.g., Kaletra), oseltamivir (Tamiflu), favipiravir, umifenovir, galidesivir, dexamethasone, colchicine, convalescent plasma, monoclonal antibodies (e.g., one or more of bamlanivimab, LY-C0VOI6, etesevimab, casirivimab, indevimab, sarilumab, tocilizumab), IL-6 inhibitors, kinase inhibitors, interferons, penicillin, vancomycin, erythromycin, azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.

[0316] Alternatively or additionally, immunogenic compositions and pharmaceutical compositions for use in accordance with the present disclosure may include, or be administered with, one or more other immunogenic compositions, pharmaceutical compositions, or therapies. Additional Components and Excipients

[0317] In addition to the antigens and the adjuvants described above, an immunogenic composition (e.g., a vaccine composition) or pharmaceutical composition may include one or more additional components.

[0318] In certain embodiments, the immunogenic composition or pharmaceutical composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.

[0319] In certain embodiments, the immunogenic composition or pharmaceutical composition includes one or more buffers such as histidine, glycine, succinate, Tris, or a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the immunogenic composition or pharmaceutical composition includes a succinate buffer. In some embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.

[0320] In certain embodiments, the immunogenic composition or pharmaceutical composition includes one or more surfactants, for example, but not limited to, polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20), Polyethylene glycol p- (l,l,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100), and 4-(l, 1,3,3- Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or non-ionic.

[0321] In certain embodiments, the immunogenic composition or pharmaceutical composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

[0322] In certain embodiments, a preservative is included in the immunogenic composition or pharmaceutical composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose immunogenic composition e.g. , vaccine) or pharmaceutical composition vials, and is less often needed in single-dose immunogenic composition e.g., vaccine) or pharmaceutical composition vials. In certain embodiments, the preservative is 2-phenoxyethanol, thimerosal, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.

Uses of Immunogenic Compositions and Pharmaceutical Compositions

[0323] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein that includes one or more antigenic polysaccharides is characterized in that one or more of the opsonization potential, or immune response to one or more antigenic polysaccharides is increased relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, one or more of the opsonization potential, immune response to the one or more antigenic polysaccharides is increased at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, the predetermined level is a pre-immune level. In some embodiments, one or more polypeptides e.g., antigenic polypeptides) are carrier proteins for one or more antigenic polysaccharides.

[0324] In some embodiments, an immunogenic composition or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polysaccharide alone. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polypeptide alone. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response.

[0325] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more strains (or variants, or serotypes) of a pathogen. In some embodiments, a pathogen is a bacterial, fungal, parasitic, and/or viral pathogen. In some embodiments, a bacterial pathogen is S. pneumoniae. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein), upon administration to a subject, induces an immune response against one or more tumors characterized by expression of such a tumor or cancer antigen.

[0326] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against .S', pneumoniae. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more serotypes of .S'. pneumoniae. In some embodiments, such an immune response may be directed against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein includes polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, such an immune response may be directed against one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein does not include polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, such an immune response may be directed against two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein (i) includes polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s); and (ii) does not include polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response against one or more serotypes of 5. pneumoniae. In some embodiments, such a protective response may be directed against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein includes polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, such a protective response may be directed against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein does not include polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, such a protective response may be directed against two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein (i) includes polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s); and (ii) does not include polysaccharide(s) present in at least one or more (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s).

[0327] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the antibody or B cell response is a memory B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the T cell response is a memory T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or a CD4-/CD8- T cell response. Tn some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies. In some embodiments, the immune response is a memory response.

[0328] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces antibody production against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polysaccharide alone. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces antibody production against one or more tumors or pathogens in the subject at level greater than a composition comprising a polypeptide antigen alone.

[0329] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polysaccharide alone. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more tumors or pathogens in the subject at a level greater than a composition comprising a polypeptide antigen alone. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response.

[0330] The immunogenic compositions (e.g., vaccines) and pharmaceutical compositions described herein may be used for prophylactic and/or therapeutic treatment of cancer or pathogen infection and/or disease. Accordingly, the present disclosure provides a method for immunizing a subject suffering from or susceptible to cancer or pathogen infection, comprising administering an immunologically effective amount of any of the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein. The subject receiving the immunization may be a male or a female, and may be an infant, child, adolescent, or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal. In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces a protective immune response against cancer or pathogen infection and/or disease.

[0331] In prophylactic embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is administered to a subject to induce an immune response that can help protect against the establishment of one or more strains (variants) of a pathogen, for example by protecting against asymptomatic infection. In some aspects, the method inhibits infection by a pathogen in an uninfected subject. In another aspect, the method may reduce transmission, replication, and/or pathogen load of one or more strains (variants) of a pathogen in a subject who is already infected.

[0332] In prophylactic embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) is administered to a subject to induce an immune response that can help protect against the establishment of one or more tumors characterized by expression of such a tumor or cancer antigen, for example by protecting against asymptomatic cancer. In some aspects, the method inhibits cancer by a tumor in an unaffected subject. In another aspect, the method may reduce metastasis, replication, and/or tumor load of one or more tumors in a subject who is already affected. [0333] In prophylactic embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae, for example by protecting against colonization, the first and necessary step in disease. In some embodiments, such an immune response may be directed against one or more (e.g., 1, 2, 3, 4,

5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein includes polysaccharide(s) present in at least one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). In some embodiments, such an immune response may be directed against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae , wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein does not include polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s) (non-vaccine types, NVTs). In some embodiments, such an immune response may be directed against two or more (e.g., 2, 3, 4, 5,

6, 7, 8, 9, 10 or more) serotypes of Streptococcus pneumoniae, wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein (i) includes polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s); and (ii) does not include polysaccharide(s) present in at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of such serotype(s). Thus, in some aspects, the method inhibits infection by S. pneumoniae in a non-colonized or uninfected subject. In another aspect, the method may reduce the duration of colonization in a subject who is already colonized.

[0334] In therapeutic embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition may be administered to a subject suffering from cancer or a pathogen infection, in an amount sufficient to treat the subject. Treating the subject, in this case, refers to reducing cancer or pathogen symptoms and/or tumor or pathogen load and/or sequelae in the subject. In some embodiments, treating the subject refers to reducing the duration of symptoms or sequelae, or reducing the intensity of symptoms or sequelae. In some embodiments, the immunogenic composition (e.g., vaccine) or pharmaceutical composition reduces transmissibility of one or more strains (variants) of a pathogen from the immunized subject to another subject. In some embodiments, the immunogenic composition (e.g., vaccine) or pharmaceutical composition reduces metastasis of one or more tumors in the immunized subject. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., relative to a control, e.g., a control subject.

[0335] In therapeutic embodiments, the immunogenic composition (e.g., vaccine) or pharmaceutical composition is administered to a subject post-tumor development or postpathogen infection. The immunogenic composition (e.g., vaccine) or pharmaceutical composition may be administered shortly after tumor development or pathogen infection, e.g. before symptoms or sequelae manifest, or may be administered during or after manifestation of symptoms or sequelae.

[0336] In some embodiments, the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein confer protective immunity, allowing an immunized subject to exhibit delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, as the result of his or her exposure to the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In certain embodiments, the reduction in severity of symptoms or sequelae is at least 25%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., relative to a control. In particular embodiments, immunized subjects may display no symptoms or sequelae upon development of a tumor or infection with a pathogen (asymptomatic cancer or infection), or do not develop a tumor or become infected by a pathogen. Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. In particular, cellular immunity may be mediated by THI or TH17 cells.

[0337] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more strains (variants) of a pathogen. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein), upon administration to a subject, induces an immune response against one or more tumors characterized by expression of such a tumor or cancer antigen.

[0338] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against S. pneumoniae. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or more) serotypes of S. pneumoniae. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against all serotypes of 5. pneumoniae comprised in such immunogenic composition (e.g., vaccine). In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response against one or more (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or more) serotypes of 5. pneumoniae. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response against all serotypes of S. pneumoniae comprised in such immunogenic composition (e.g., vaccine).

[0339] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the antibody or B cell response is a memory B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the T cell response is a memory T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or CD4-/CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies. In some embodiments, the immune response is a memory response.

[0340] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an antibody or B cell response against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polysaccharide alone. In some embodiments, an immunogenic composition e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an antibody or B cell response against one or more tumors or pathogens in the subject at level greater than a composition comprising a polypeptide antigen alone. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0341] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a T cell response against one or more tumors or pathogens in the subject at a level greater than a composition comprising an antigenic polysaccharide alone. In some embodiments, an immunogenic composition (e.g. , vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a T cell response against one or more tumors or pathogens in the subject at level greater than a composition comprising a polypeptide antigen alone. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0342] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents disease due to infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents one or more symptoms due to infection by one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastro-intestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents one or more of Invasive Pneumococcal Disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis due to infection by S. pneumoniae.

[0343] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of disease due to infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of one or more symptoms due to infection by one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastrointestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence of one or more of Invasive Pneumococcal Disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis due to infection by S. pneumoniae.

[0344] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of disease due to infection by one or more strains (variants) of a pathogen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of one or more symptoms due to infection by one or more strains (variants) of a pathogen. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastro-intestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of one or more of Invasive Pneumococcal Disease (IPD), bacteremia, sepsis, organ damage, meningitis, pneumonia, otitis media, and sinusitis due to infection by S. pneumoniae.

[0345] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) treats or prevents cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) treats or prevents cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, a tumor or cancer antigen polypeptide (e.g., as described herein) present in an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein to be administered to a cancer subject is identified to be present in the subject’s tumor or cancer cells. [0346] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen.

[0347] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is useful to slow down the progression of cancer or growth of a tumor. In some embodiments, In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen.

[0348] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits transmission of one or more strains (variants) of a pathogen from the subject to another subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits asymptomatic infection by one or more strains (variants) of a pathogen in the subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits replication and/or reduces pathogen load of one or more strains (variants) of a pathogen in the subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits colonization by one or more strains (variants) of a pathogen in the subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits colonization by one or more strains (variants) of a pathogen of a mucosal surface of the subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits colonization by one or more strains (variants) of a pathogen in the nasopharynx of the subject.

[0349] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits metastasis of one or more tumors characterized by expression of such a tumor or cancer. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits asymptomatic cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits replication and/or reduces tumor load of one or more tumors characterized by expression of such a tumor or cancer antigen in the subject.

[0350] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more tumors or pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a protective immune response against one or more tumors or pathogens in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant). In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0351] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits immunogenicity non-inferior to (e.g., immunogenicity at least as effective as) that elicited by administration of a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits immunogenicity non-inferior to (e.g., immunogenicity at least as effective as) that elicited by administration of a control composition against one or more tumors or pathogens. In some embodiments, the immunogenicity is determined by measuring the amount of anti-polysaccharide antibody of one or more polysaccharides present in the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In some embodiments, the control composition may be PCV 13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant). In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0352] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits immunogenicity superior to that elicited by administration of a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits immunogenicity superior to that elicited by administration of a control composition against one or more tumors or pathogens. In some embodiments, the immunogenicity is determined by measuring the amount of antipolysaccharide antibody of one or more polysaccharides present in the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant). In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0353] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer non-inferior to (e.g., a functional antibody titer at least as effective as) that elicited by administration of a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer non-inferior to (e.g., a functional antibody titer at least as effective as) that elicited by administration of a control composition against one or more tumors or pathogens. In some embodiments, the functional antibody titer is measured by an opsonophagocytic assay (e.g., ones as described herein). In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer non-inferior to (e.g., a functional antibody titer at least as effective as) that elicited by administration of a control composition against one or more tumors or pathogens, wherein such an immunogenic composition (e.g., vaccine) or pharmaceutical composition does not include polysaccharide(s) present in at least one of the tumors or pathogens (non-vaccine types, NVTs). In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant). In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0354] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer suprior to that elicited by administration of a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer suprior to that elicited by administration of a control composition against one or more tumors or pathogens. In some embodiments, the functional antibody titer is measured by an opsonophagocytic assay (e.g. , ones as described herein). In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, elicits a functional antibody titer suprior to that elicited by administration of a control composition against one or more tumors or pathogens, wherein such an immunogenic composition (e.g., vaccine) or pharmaceutical composition does not include polysaccharide(s) present in at least one of the tumors or pathogens (non-vaccine types, NVTs). In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant). In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0355] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an immune response against one or more strains (variants) of a pathogen at a level greater than a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein), upon administration to a subject, induces an immune response against one or more tumors characterized by expression of such a tumor or cancer antigen at a level greater than a control composition. In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or CD4-/CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0356] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces an antibody or B cell response against one or more tumors or pathogens in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0357] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a T cell response against one or more tumors or pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein, upon administration to a subject, induces a T cell response against one or more tumors or pathogens in the subject at level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0358] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents disease due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein treats or prevents one or more symptoms due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastro-intestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0359] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of disease due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits or reduces the rate of occurrence or recurrence of one or more symptoms due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastro-intestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0360] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of disease due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein reduces the severity of one or more symptoms due to infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, the one or more symptoms include pneumonia, organ damage, upper respiratory symptoms, gastro-intestinal symptoms, neurological symptoms, myocarditis, inflammation, fever, chills, fatigue, headache, nausea, muscle or body ache, shortness of breath or difficulty breathing, loss of sense of smell (hyposmia, anosmia), loss of sense of taste (hypogeusia, ageusia), and/or multi-inflammatory syndrome of children or adults (MIS-C, MIS-A). In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0361] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) treats or prevents cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) treats or prevents disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) treats or prevents one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0362] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits or reduces the rate of occurrence or recurrence of one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. Tn some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0363] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of disease due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) reduces the severity of one or more symptoms due to cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0364] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits transmission of one or more strains (variants) of a pathogen from the subject to another subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits colonization by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits colonization by one or more strains (variants) of a pathogen in the nasopharynx of the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits asymptomatic infection by one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein inhibits replication and/or reduces pathogen load of one or more strains (variants) of a pathogen in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition. In some embodiments, the control composition may be PCV13, PCV20, or PPSV23. In some embodients, the control composition may be a composition comprising the same immunogenic composition formulated with a higher concentration of an adjuvant (e.g., an aluminum-based adjuvant).

[0365] In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits metastasis of one or more tumors characterized by expression of such a tumor or cancer antigen at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits asymptomatic cancer by one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, upon administration to a subject, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) inhibits replication and/or reduces tumor load of one or more tumors characterized by expression of such a tumor or cancer antigen in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0366] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is administered to a subject between about 6 weeks and about 5 years (e.g., prior to the 6^th birthday) for active immunization for the prevention of disease caused by one or more strains (variants) of a pathogen. In some embodiments, such one or more strains (variants) of a pathogen are selected from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) is administered to a subject between about 6 weeks and about 5 years (e.g., prior to the 6^th birthday) for active immunization for the prevention of disease caused by one or more tumors characterized by expression of such a tumor or cancer antigen.

[0367] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is administered to a subject between about 6 years and about 17 years (e.g., prior to the 18^th birthday) for active immunization for the prevention of disease caused by one or more strains (variants) of a pathogen. In some embodiments, such one or more strains (variants) of a pathogen are selected from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23 A, 23B, 23F, 24F, 31, 33F, 35B, and 38. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) is administered to a subject between about 6 years to about 17 years e.g., prior to the 18^th birthday) for active immunization for the prevention of disease caused by one or more tumors characterized by expression of such a tumor or cancer antigen.

[0368] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein is administered to a subject 18 years or older for active immunization for the prevention of disease caused by one or more strains (variants) of a pathogen. In some embodiments, such one or more strains (variants) of a pathogen are selected from Streptococcus pneumoniae serotypes 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, an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein comprising a tumor antigen polypeptide (e.g., as described herein) is administered to a subject 18 years or older for active immunization for the prevention of disease caused by one or more tumors characterized by expression of such a tumor or cancer antigen.

Antibody Compositions

[0369] Some embodiments provide for an antibody composition comprising antibodies raised in a mammal immunized with an immunogenic complex or immunogenic composition of the invention. In some embodiments, an antibody comprises at least one antibody selected from the group consisting of mAbs and anti-idiotype antibodies. In some embodiments, an antibody composition comprises neutralizing antibodies. In some embodiments, an antibody composition comprises an isolated gamma globulin fraction. In some embodiments, an antibody composition comprises polyclonal antibodies. In some embodiments, the antibody composition is administered to a subject. In some embodiments, the antibody composition administered to a subject confers passive immunization. Methods of Administration

[0370] In some embodiments, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein are administered to a subject (e.g. an infant, a toddler, a juvenile, or an older adult) at risk of developing disease due to one or more tumors or pathogens. In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age or older. In some embodiments, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein are administered to a subject at elevated risk of developing a disease due to one or more tumors or pathogens, e.g. , immunocompromised subjects, subjects having sickle cell disease or other hemoglobinopathies, congenital or acquired asplenia, splenic dysfunction, chronic renal failure or nephrotic syndrome, diseases associated with treatment with immunosuppressive drugs or radiation therapy (including malignant neoplasm, leukemia, lymphomas, Hodgkin's disease, or solid organ transplantation), congenital or acquired immunodeficiency, HIV infection, cerebrospinal fluid leaks, cochlear implant(s), chronic heart disease, chronic lung disease, diabetes mellitus, alcoholism, chronic liver disease, cigarette smoking, asthma, generalized malignancy, multiple myeloma, or solid organ transplantation. It will be appreciated that a subject can be considered at risk for developing a disease without having been diagnosed with any symptoms of the disease. For example, if the subject is known to have been, or to be intended to be, in situations with relatively high risk of infection, that subject will be considered at risk for developing the disease.

[0371] Any effective route of administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein may be utilized such as, for example, oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, transdermal, intradermal, rectal, vaginal, topical, ocular, pulmonary, or by contact application. In some embodiments, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions may be injected e.g., via intramuscular, intraperitoneal, intradermal and/or subcutaneous routes); or delivered via the mucosa (e.g., to the oral/alimentary, respiratory, and/or genitourinary tracts). In some embodiments, it may be desirable to administer different doses of an immunogenic composition (e.g., vaccine) or pharmaceutical composition by different routes; in some embodiments, it may be desirable to administer different components of one dose via different routes. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein is administered intramuscularly. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein is administered subcutaneously.

[0372] In some embodiments, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions are administered intradermally. Conventional technique of intradermal injection, the "Mantoux procedure", comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26-31 gauge) facing upwards the needle is inserted at an angle of between 10-15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced while providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.

[0373] Devices that are specifically designed to administer liquid agents into or across the skin have been described, for example the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described for example in WO 01/13977; US Patent No. 5,480,381, US Patent No. 5,599,302, US Patent No. 5,334,144, US Patent No. 5,993,412, US Patent No. 5,649,912, US Patent No. 5,569,189, US Patent No. 5,704,911, US Patent No. 5,383,851 , US Patent No. 5,893,397, US Patent No. 5,466,220, US Patent No. 5,339,163, US Patent No. 5,312,335, US Patent No. 5,503,627, US Patent No. 5,064,413, US Patent No.

5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US Patent No. 4,941,880, US Patent No. 4,940,460, WO 97/37705 and WO 97/13537. Other methods of intradermal administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or applied to the surface of the skin (transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).

[0374] As described above, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions may be administered as a single dose or as multiple doses. It will be appreciated that an administration is a single “dose” so long as all relevant components are administered to a subject within a window of time; it is not necessary that every component be present in a single composition. For example, administration of two different immunogenic compositions (e.g., vaccines) or pharmaceutical compositions, within a period of less than 24 h, is considered a single dose. To give but one example, immunogenic compositions (e.g., vaccines) or pharmaceutical compositions having different antigenic components may be administered in separate compositions, but as part of a single dose. As noted above, such separate compositions may be administered via different routes or via the same route. Alternatively or additionally, in embodiments wherein an immunogenic composition (e.g., vaccine) or pharmaceutical composition comprises a combination of immunogenic compositions and additional types of active agents, immunogenic compositions may be administered via one route, and an additional active agent may be administered by the same route or by a different route.

[0375] Immunogenic compositions (e.g., vaccines) and pharmaceutical compositions are administered in such amounts and for such time as is necessary to achieve a desired result. In certain embodiments of the present invention, an immunogenic composition (e.g., vaccine) or pharmaceutical composition comprises an immunologically effective amount of at least immunogenic composition. The exact amount required to achieve an immunologically effective amount may vary, depending on the immunogenic composition, and from subject to subject, depending on the species, age, and general condition of the subject, the stage of the disease, the particular pharmaceutical mixture, its mode of administration, and the like.

[0376] The amount of polypeptide(s) (e.g., antigenic polypeptide(s)), polymer(s) (e.g., antigenic polysaccharide(s)), or conjugate(s) in each immunogenic composition (e.g. , vaccine) or pharmaceutical composition dose is selected to allow the composition, when administered as described herein, to induce an appropriate immune-protective response without significant, adverse side effects.

[0377] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein induces a Tnl and/or TH17 cell response upon administration to a subject. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein induces an opsonic/bactericidal response against S. pneumoniae upon administration to a subject. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein reduces rate of transmission and/or colonization by S. pneumoniae upon administration to a subject. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein reduces rate of transmission and/or colonization of the mucosal surfaces by .S'. pneumoniae upon administration to a subject.

[0378] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein reduces rate of transmission and/or colonization of the nasopharynx or the lungs by S. pneumoniae upon transmission. In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein inhibits or reduces asymptomatic infection by .S', pneumoniae upon administration to a subject.

[0379] Some embodiments provide for a method of immunizing a subject against S. pneumoniae infection comprising administering to the subject an immunologically effective amount of an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein. Some embodiments provide for a method of immunizing a subject against .S', pneumoniae infection comprising administering to the subject an immunologically effective amount of an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein.

[0380] Some embodiments provide an immunogenic composition (e.g., vaccine) or pharmaceutical composition as described herein for use in therapy. For example, an immunogenic composition (e.g., vaccine) or pharmaceutical composition as described herein for use in the treatment or prevention of disease due to one or more tumors or pathogens in a subject such as a human subject. Some embodiments provide an immunogenic composition (e.g., vaccine) or pharmaceutical composition as described herein for use in the treatment or prevention of disease due to one or more tumors or pathogens in a subject such as a human subject. Further embodiments provide a use of an immunogenic composition (e.g., vaccine) or pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prevention of disease due to one or more tumors or pathogens. In some embdiments, such a disease is due to infection and/or colonization by Streptococcus pneumoniae. The characteristics of methods of treatment or prevention disclosed herein are equally applicable to an immunogenic composition (e.g., vaccine) or pharmaceutical composition for use, or a use of an immunogenic composition (e.g., vaccine) or pharmaceutical composition, in the manufacture of a medicament. Combination Prophylaxis or Combination Therapy

[0381] In some embodiments, an immunogenic composition (e.g., vaccine) or pharmaceutical composition disclosed herein may be administered in combination with one or more additional agents. In some embodiments, an additional agent may be or comprise a therapeutic agent. In some embodiments, such a therapeutic agent may be or comprise an antibacterial agent. In some embodiments, such a therapeutic agent may be or comprise an anti-cancer agent. In some embodiments, an additional agent may be or comprise one or more additional vaccines. In some embodiments, an additional agent may be or comprise PCV 13. In some embodiments, an additional agent may be or comprise PCV20. In some embodiments, an additional agent may be or comprise PPSV23. In some embodiments, an additional agent may be or comprise an antibiotic.

Dosing

[0382] In some embodiments, administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition described herein may involve the delivery of a single dose. In some embodiments, administration may involve an initial dose followed by one or several additional immunization doses, adequately spaced. An immunization schedule or regimen is a program for the administration of one or more specified doses of one or more specified immunogenic compositions (e.g., vaccines) or pharmaceutical compositions, by one or more specified routes of administration, at one or more specified ages of a subject.

[0383] Immunization schedules of the present disclosure are provided to induce an immune response e.g., an immunoprotective response) in a subject sufficient to reduce at least one measure selected from the group consisting of incidence, prevalence, frequency, and/or severity of at least one infection, disease, or disorder, and/or at least one surrogate marker of the infection, disease, or disorder, in a population and/or subpopulation of the subject(s). A supplemental immunization schedule is one which has this effect relative to the standard schedule which it supplements. A supplemental schedule may call for additional administrations and/or supra-immunogenic doses of the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions disclosed herein, found in the standard schedule, or for the administration of immunogenic compositions (e.g., vaccines) or pharmaceutical compositions not part of the standard schedule. A full immunization schedule of the present invention may comprise both a standard schedule and a supplemental schedule. Exemplary sample immunization schedules are provided for illustrative purposes. Detailed descriptions of methods to assess immunogenic response discussed herein allow one to develop alterations to the sample immunization schedules without undue experimentation.

[0384] In some embodiments of the present disclosure, a first administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is more than about 2 weeks old, more than about 5 weeks old, more than about 1 year old, more than about 2 years old, more than about 15 years old, or more than about 18 years old.

[0385] In some embodiments, a first administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is about two months old. In some embodiments, a second administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is about four months old. In some embodiments, a third administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is about six months old. In some embodiments, a fourth administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is between about twelve months old and about fifteen months old.

[0386] In some embodiments of the present disclosure, a first administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition occurs when a subject is more than about 18 years old, more than about 50 years old, more than about 55 years old, more than about 60 years old, more than about 65 years old, or more than about 70 years old.

[0387] In some embodiments of the disclosure, a single administration of an immunogenic composition (e.g., vaccine) or pharmaceutical composition is employed. It is possible that the purposes of the present disclosure can be served with a single administration, especially when one or more utilized polypeptide(s) (e.g., antigenic polypeptide(s)), polymers (e.g., polysaccharide(s)), and/or immunogenic complex(es) or combinations thereof is/are strong, and in such a situation a single dose schedule is sufficient to induce a lasting immune-protective response.

[0388] In certain embodiments, it is desirable to administer two or more doses of an immunogenic composition (e.g., vaccine) or pharmaceutical composition, for greater immune-protective efficacy and coverage. Thus, in some embodiments, a number of doses is at least two, at least three or more doses. There is no set maximum number of doses; however, it is good clinical practice not to immunize more often than necessary to achieve the desired effect.

[0389] Without being bound by theory, a first dose of an immunogenic composition (e.g., vaccine) or pharmaceutical composition administered according to the disclosure may be considered a “priming” dose. In certain embodiments, more than one dose is included in an immunization schedule. In such a scenario, a subsequent dose may be considered a “boosting” dose.

[0390] A priming dose may be administered to a naive subject (a subject who has never previously received an immunogenic composition (e.g., vaccine) or pharmaceutical composition). In some embodiments, a priming dose may be administered to a subject who has previously received an immunogenic composition (e.g., vaccine) or pharmaceutical composition at least five or more years previous to administration of an initial immunogenic composition (e.g., vaccine) or pharmaceutical composition dose according to the invention. In other embodiments, a priming dose may be administered to a subject who has previously received an immunogenic composition (e.g., vaccine) or pharmaceutical composition at least twenty or more years previous to administration of a priming immunogenic composition (e.g., vaccine) or pharmaceutical composition according to the disclosure.

[0391] When an immunization schedule calls for two or more separate doses, the interval between doses is considered. The interval between two successive doses may be the same throughout an immunization schedule, or it may change as the subject ages. In immunization schedules of the present disclosure, once a first immunogenic composition (e.g., vaccine) or pharmaceutical composition dose has been administered, there is a first interval before administration of a subsequent dose. A first interval is generally at least about 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 12 months, or longer. Where more than one subsequent dose(s) are administered, second (or higher) intervals may be provided between such subsequent doses. In some embodiments, all intervals between subsequent doses are of the same length; in other embodiments, second intervals may vary in length. In some embodiments, the interval between subsequent doses may be at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months or at least about 2 years. In certain embodiments, the interval between doses may be up to 3 years, up to about 4 years, or up to about 5 years or 10 years or more. In certain embodiments, intervals between subsequent doses may decrease as the subject ages.

[0392] It will be appreciated by those skilled in the art that a variety of possible combinations and sub-combinations of the various conditions of timing of the first administration, shortest interval, largest interval and total number of administrations (in absolute terms, or within a stated period) exist, and all of these combinations and subcombinations should be considered to be within the inventor's contemplation though not explicitly enumerated here.

Exemplary Assays for Determining Immune Response

[0393] In some embodiments, a method of assessing the immunogenicity of an immunogenic composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by opsonophagocytic killing (OPK, OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the immunogenic composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the immunogenic composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer (e.g., an antigenic polysaccharide) present in the immunogenic composition and not comprise a polypeptide (e.g. , an antigenic polypeptide) present in the immunogenic composition. In some embodiments, a control composition may comprise a polypeptide (e.g. , an antigenic polypeptide) present in the immunogenic composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the immunogenic composition. In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition, and not comprise a polymer e.g. , an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the immunogenic composition.

[0394] In some embodiments, a method of assessing the potency of an immunogenic composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by OPK (OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the immunogenic composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the immunogenic composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer (e.g., an antigenic polysaccharide) present in the immunogenic composition and not comprise a polypeptide (e.g. , an antigenic polypeptide) present in the immunogenic composition. In some embodiments, a control composition may comprise a polypeptide (e.g. , an antigenic polypeptide) present in the immunogenic composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the immunogenic composition. In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition, and not comprise a polymer (e.g. , an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the immunogenic composition.

[0395] In some embodiments, a method of assessing the immunogenicity of an vaccine composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by opsonophagocytic killing (OPK, OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the vaccine composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the vaccine composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer (e.g., an antigenic polysaccharide) present in the vaccine composition and not comprise a polypeptide (e.g., an antigenic polypeptide) present in the vaccine composition. In some embodiments, a control composition may comprise a polypeptide (e.g., an antigenic polypeptide) present in the vaccine composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the vaccine composition. In some embodiments, a control composition may comprise an adjuvant present in the vaccine composition, and not comprise a polymer (e.g. , an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the vaccine composition.

[0396] In some embodiments, a method of assessing the potency of an vaccine composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by OPK (OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the vaccine composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the vaccine composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer (e.g., an antigenic polysaccharide) present in the vaccine composition and not comprise a polypeptide (e.g., an antigenic polypeptide) present in the vaccine composition. In some embodiments, a control composition may comprise a polypeptide (e.g. , an antigenic polypeptide) present in the vaccine composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the vaccine composition. In some embodiments, a control composition may comprise an adjuvant present in the vaccine composition, and not comprise a polymer (e.g., an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the vaccine composition.

[0397] In some embodiments, a method of assessing the immunogenicity of an pharmaceutical composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by opsonophagocytic killing (OPK, OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the pharmaceutical composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the pharmaceutical composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer (e.g., an antigenic polysaccharide) present in the pharmaceutical composition and not comprise a polypeptide (e.g., an antigenic polypeptide) present in the pharmaceutical composition. In some embodiments, a control composition may comprise a polypeptide (e.g. , an antigenic polypeptide) present in the pharmaceutical composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the pharmaceutical composition. In some embodiments, a control composition may comprise an adjuvant present in the pharmaceutical composition, and not comprise a polymer (e.g., an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the pharmaceutical composition.

[0398] In some embodiments, a method of assessing the potency of an pharmaceutical composition described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by OPK (OPA), plaque reduction neutralization test (PRNT), serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the pharmaceutical composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the pharmaceutical composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise a polymer e.g., an antigenic polysaccharide) present in the pharmaceutical composition and not comprise a polypeptide (e.g., an antigenic polypeptide) present in the pharmaceutical composition. In some embodiments, a control composition may comprise a polypeptide (e.g. , an antigenic polypeptide) present in the pharmaceutical composition and not comprise a polymer (e.g., an antigenic polysaccharide) present in the pharmaceutical composition. In some embodiments, a control composition may comprise an adjuvant present in the pharmaceutical composition, and not comprise a polymer (e.g., an antigenic polysaccharide) and/or a polypeptide (e.g., an antigenic polypeptide) present in the pharmaceutical composition.

[0399] In some embodiments, a method of assessing the immunogenicity and/or potency of an immunogenic complex comprises evaluating an immune response to immunogenic or vaccine compositions comprising one or more immunogenic complexes. In some embodiments, the method of assessing the immunogenicity and/or potency of an immunogenic complex described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by OPK, plaque reduction neutralization test (PRNT), serum bactericidal killing (SB A), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the immunogenic composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the immunogenic composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition.

[0400] Generally speaking, it may be desirable to assess humoral responses, cellular responses, and/or interactions between the two. Where humoral responses are being assessed, antibody titers and/or types (e.g., total IgG, IgGl, IgG2, IgM, IgA, etc.) to specific pathogen polysaccharides or polypeptides (either serotype-specific or conserved across two or more serotypes) may be determined, for example before and/or after administration of an initial or a boosting dose of vaccine (and/or as compared with antibody levels in the absence of antigenic stimulation). Cellular responses may be assessed by monitoring reactions such as delayed type hypersensitivity responses, etc. to the carrier protein. Cellular responses can also be measured directly by evaluating the response of peripheral blood mononuclear cells (PBMCs) monocytes to stimulation with the antigens of interest. Precursor and memory B cell populations may be assessed in enzyme linked immunospot (ELISpot) assays directed against specific pathogen polysaccharides or polypeptides.

[0401] Any of a variety of assays may be employed to detect levels and/or activity of antibodies in subject sera. Suitable assays include, for example, ligand binding assays, such as radioimmunoassay (RIAs), ELISAs, and multiplex assays (Luminex, Bioplex, MSD); functional assays, such as opsonophagocytic assays or internalization assays; and in vivo assays in animal models of disease. In some embodiments, where the disease is a viral disease, parameters of in vivo assays include viral clearance, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of a virus that are the targets of the immunogenic composition. In some embodiments, where the disease is a bacterial disease, parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, inhibition or reduction of asymptomatic infection, reduction of mortality, and passive and active protection following challenge with one or more strains (variants) of bacteria that are the targets of the immunogenic composition. In some embodiments, where the disease is a cancer or tumor, parameters of in vivo assays include reduction in tumor size and/or number, reduction or prevention of tumor metastasis, inhibition or reduction of asymptomatic cancer, reduction in mortality, and passive and active protection following challenge with one or more tumors characterized by expression of one or more tumor or cancer antigens that are the targets of the immunogenic composition.

[0402] The RIA method detects specific antibodies through incubation of sera with radio-labeled polysaccharides or polypeptides in suspension (e.g., Schiffiman et al, 1980). The antigen- antibody complexes are then precipitated with ammonium sulfate and the radiolabeled pellets assayed for counts per minute (cpm).

[0403] In the ELISA detection method, specific antibodies from the sera of vaccinated subjects are quantitated by incubation with polysaccharides or polypeptides (either serotype-specific or conserved across two or more serotypes) which have been adsorbed to a solid support (e.g. , Koskela and Leinonen, 1981; Kojima et al., 1990; Concepcion and Frasch, 2001). The bound antibody is detected using enzyme-conjugated secondary detection antibodies. The ELISA also allows isotyping and subclassing of the immune response (z.<?., IgM vs. IgG or IgGl vs. IgG2) by using isotype- or subclass-specific secondary antibodies and can be adapted to evaluate the avidity of the antibodies (Anttila et al, 1998; Romero- Steiner et al, 2005). Multiplex assays (e.g., Luminex) facilitate simultaneous detection of antibodies to multiple antigens. Capsular polysaccharide(s) or polypeptides are conjugated to spectrally distinct microspheres that are mixed and incubated with serum. The antibodies bound to the polysaccharides or polypeptides on the coated microspheres are detected using a secondary antibody (e.g., R-Phycoerythrin-conjugated goat anti-human IgG).

[0404] An approach for assessing functional antibody in serum is an opsonophagocytic assay (OP A) or a concentrated opsonophagocytic assay (COP A), which quantitates only the antibodies that can opsonize the bacteria, leading to ingestion and killing of the bacteria. The standard assay utilizes a human phagocytic effector cell, a source of complement, bacteria, and diluted sera. The assay readout is the serum endpoint titer at which there is >50% killing compared to bacteria incubated with complement and human cells alone (Romero-Steiner et al., 1997). This killing OPA can also be multiplexed by utilizing target strains of pathogen that carry different antibiotic resistance markers (Kim et al., 2003). Another type of multiplex opsonic assay is a nonkilling assay in which the uptake by phagocytic effector cells of fluorescent stained encapsulated pathogen or fluorescent microspheres conjugated with antigenic polysaccharides or polypeptides from a target pathogen in the presence of diluted sera plus a complement source is evaluated by flow cytometry (Martinez et al., 1999). Opsonic activity of serum antibody plus complement can also be evaluated by measuring the oxidative response of phagocytic human effector cells to ingested pathogen (Munro et al., 1985; Ojo-Amaize et al., 1995).

[0405] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by vaccines of the present disclosure. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which provides protection against pneumonia, bacteremia, colonization of organs or tissues, or mortality is determined (Stack et al., 1998; Saeland et al., 2000).

[0406] In some embodiments, efficacy of immunization may be determined by assaying one or more cytokine levels by stimulating T cells from a subject after immunization. The one or more cytokine levels may be compared to the one or more cytokine levels in the same subject before immunization. Increased levels of the one or more cytokine, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase over pre-immunization cytokine levels, would indicate an increased response to the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In some embodiments, the one or more cytokines are selected from GM-CSP; IL-la; IL-1P; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL- 12; IL-17A, IL-17F or other members of the IL-17 family; IL-22; IL-23; IFN- a; IFN-P; IFN-y; MIP-la; MIP-1P; TGF-P; TNFa, or TNF-p. In a non-limiting example, efficacy of immunization may be determined by assaying IL-17 levels (particularly IL-17A) by stimulating T cells from a subject after immunization. The IL-17 levels may be compared to IL-17 levels in the same subject before immunization. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the immunogenic composition (e.g., vaccine) or pharmaceutical composition.

[0407] In some embodiments, one may assay neutrophils in the presence of T cells or antibodies from the patient for pathogen (e.g. , viral or bacterial) or tumor killing. Increased pathogen (e.g. , viral or bacterial) or tumor killing, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the immunogenic composition (e.g., vaccine) or pharmaceutical composition. For example, one may measure TH17 cell activation, where increased TH17 cell activation, such as a 1.5 fold, 2- fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In another non-limiting example, one may measure Tnl cell activation, where increased THI cell activation, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the immunogenic composition (e.g., vaccine) or pharmaceutical composition. One may also measure levels of an antibody specific to the vaccine, where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased immunogenic composition (e.g., vaccine) or pharmaceutical composition efficacy. In certain embodiments, two or more of these assays are used. For example, one may measure IL-17 levels and the levels of immunogenic composition (e.g., vaccine) or pharmaceutical composition -specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of pathogen (e.g. , viral or bacterial) infection or tumor development in vaccinated individuals compared to unvaccinated individuals.

[0408] Immunogenic composition (e.g., vaccine) or pharmaceutical composition efficacy may also be assayed in various model systems such as the mouse challenge model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering the test immunogenic composition (e.g., vaccine) or pharmaceutical composition to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of pathogen or tumor. In some cases, a challenge dose administered intranasally or intratracheally is sufficient to cause pathogen infection and/or a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered intranasally is sufficient to cause pathogen colonization (especially nasal colonization) in an unvaccinated animal, and in some cases a challenge dose administered via aspiration is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intraperitoneal injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intravenous injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intraperitoneal injection is sufficient to cause tumor development and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intravenous injection is sufficient to cause tumor development and a high rate of lethality in unvaccinated animals. One can then measure the reduction in infection, the reduction in colonization, reduction in tumor development, or the reduction in lethality in vaccinated animals.

[0409] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by immunogenic compositions (e.g., vaccines) or pharmaceutical compositions of the present disclosure. In such passive protection systems, mice or rats are challenged with the pathogen or the tumor plus diluted sera, and the endpoint titer of the sera which provides protection against bacteremia, colonization of organs or tissues, tumor development, or mortality is determined (see, e.g., Stack et al. 1998; Saeland et al. 2000).

Kits or Delivery Devices

[0410] The present disclosure also provides for kits for producing an immunogenic complex as disclosed herein which is useful for an investigator to tailor an immunogenic complex with their preferred polymer (e.g. , an antigenic polysaccharide) and polypeptide (e.g., an antigenic polypeptide), e.g., for research purposes to assess the effect of an antigen, or a combination of antigens on immune response. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: a container comprising a polymer (e.g., an antigenic polysaccharide) cross-linked with a plurality of first affinity molecules; a container comprising a complementary affinity molecule which associates with the first affinity molecule, wherein the complementary affinity molecule associates with a polypeptide (e.g., an antigenic polypeptide or a carrier protein); a container comprising an antigenic polypeptide; a container comprising a carrier protein; a container comprising a fusion protein; a container comprising an antigenic polypeptide associated with a complementary affinity molecule; a container comprising a fusion protein associated with a complementary affinity molecule.

[0411] In another embodiment, the kit comprises a container comprising a polymer (e.g., a polysaccharide); a container comprising a plurality of first affinity molecules; and a container comprising a cross-linking reagent for cross-linking the first affinity molecules to the polysaccharide, for example, but not limited to, CDAP (l-cyano-4- dimethylaminopyridinium tetrafluoroborate), and EDC (l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride).

[0412] In another embodiment, the kit comprises a container comprising a polypeptide (e.g., an antigenic polypeptide or carrier protein), and a container comprising a complementary affinity molecule, which associates with a first affinity molecule. In some embodiments, the kit further comprises a means to attach the complementary affinity molecule to the polypeptide (e.g., the antigenic polypeptide or carrier protein), where the means can be by a cross-linking reagent or by some intermediary protein.

[0413] In some embodiments, the kit can comprise at least one co- stimulation factor, which can be added to the polymer (e.g. , a polysaccharide) or another polymer. In some embodiments, the kit comprises a cross-linking reagent, for example, but not limited to, CDAP (l-cyano-4- dimethylaminopyridinium tetrafluoroborate); EDC (l-Ethyl-3-| 3- dimethylaminopropyl] carbodiimide hydrochloride); sodium cyanoborohydride; cyanogen bromide; and ammonium bicarbonate/iodoacetic acid, for linking the co-factor to the polymer (e.g., a polysaccharide) or to another polymer. [0414] A variety of kits and components can be prepared for use in the methods described herein, depending upon the intended use of the kit, the particular target polymer (e.g., antigenic polysaccharide) and polypeptide (e.g., antigenic polypeptide) and the needs of the user.

[0415] In some embodiments, a kit can further include informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of compositions described herein. The informational material of a kit is not limited in its form. In some instances, the informational material can include information about production of a composition described herein, amino acid sequence of a polypeptide described herein, nucleic acid sequence encoding a polypeptide described herein, molecular weight of composition described herein, concentration, date of expiration, batch or production site information, and so forth.

[0416] In some cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. The informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In other instances, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a composition described herein and/or their use in the methods described herein. The informational material can also be provided in any combination of formats.

[0417] In addition to the components described above, a kit can include other ingredients, such as a solvent or buffer, a salt, a surfactant, a stabilizer, or a preservative. A kit can also include other agents, e.g., a second or third agent. The components can be provided in any form, e.g. , liquid, dried or lyophilized form. The components can be substantially pure (although they can be combined together or delivered separate from one another) and/or sterile. When the components are provided in a liquid solution, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. When the components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

[0418] The present disclosure also provides for containers (e.g., vials and syringes) containing immunogenic compositions (e.g., vaccines) or pharmaceutical compositions described herein. In some embodiments, such immunogenic compositions (e.g., vaccines) or pharmaceutical compositions are formulated with other ingredients such as a solvent or buffer, a salt, a surfactant, a stabilizer, or a preservative. In some embodiments where the container is a vial, the components can be provided in liquid, or dried or lyphoilized form. In some embodiments, where the container is a syringe the components can be provided in liquid form. When the components are provided in a liquid solution, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. When the components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit. In some embodiments, a container includes a single dose of the immunogenic composition (e.g., vaccine) or pharmaceutical composition (e.g., a syringe or vial containing a single dose). In some embodiments, a container that includes a single dose may include an overfill sufficient to deliver a single dose of the immunogenic composition (e.g., vaccine) or pharmaceutical composition. In some embodiments, a container includes multiple doses of the immunogenic composition (e.g., vaccine) or pharmaceutical composition (e.g., a vial containing multiple doses).

Exemplary Embodiments

[0419] Exemplary embodiments as described below are also within the scope of the present disclosure:

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 embodiment 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 embodiments 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 embodiments 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 embodiment 5, wherein the immune response is or comprises an antibody and/or B cell response.

7. The pharmaceutical composition or method of embodiment 5 or embodiment 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 embodiments 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 embodiments 4-8, wherein the reference composition is formulated for administration to a human.

10. The pharmaceutical composition or method of any one of embodiments 1-9, wherein the pharmaceutical composition is formulated for administration to a human. 11. The pharmaceutical composition or method of any one of embodiments 1-10, wherein the concentration of aluminum in the pharmaceutical composition is about 0.125 mg/mL to about 1.25 mg/mL.

12. The pharmaceutical composition or method of any one of embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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, 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. 19. The pharmaceutical composition or method of any one of embodiments 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: 1

20. The pharmaceutical composition or method of any one of embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiment 33, wherein the aluminum-based adjuvant is aluminum phosphate. 35. The pharmaceutical composition or method of embodiment 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 embodiment 34 or embodiment 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiment 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 embodiments 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 embodiments 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, 7 A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, HA, 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, 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.

44. The pharmaceutical composition or method of any one of embodiments 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, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

45. The pharmaceutical composition or method of any one of embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiment 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 embodiments 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 embodiment 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 embodiments 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 embodiment 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 embodiments 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; and 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, 11A, 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.;

57. The pharmaceutical composition or method of any one of embodiments 1-56, wherein the biotin-binding moiety is or comprises a rhizavidin polypeptide.

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

60. The pharmaceutical composition or method of any one of embodiments 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 embodiments 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 embodiments 1, 2, and 4-61.

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

64. The dose of embodiment 62 or embodiment 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 embodiments 62-64, wherein the amount of aluminum in the dose is about 0.25 mg.

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

67. The dose of any one of embodiments 62-66, wherein the amount of aluminum in the dose is at least 0.125 mg. 68. The dose of any one of embodiments 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 embodiments 1, 2, 4-61 or the dose of any one of embodiments 62-68.

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

73. The method of embodiment 71 or embodiments 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 embodiment 73, wherein the immune response is or comprises an antibody and/or B cell response.

75. The method of embodiment 73 or embodiment 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 embodiment 75, wherein the immune response is at a level that is at least 20% higher than the corresponding reference level.

77. The method of embodiment 75 or embodiment 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 embodiments 71-77, wherein the subject is a human subject.

Exemplification

Example 1: Preparation of Exemplary Fusion Proteins CPI and SPP2

Vector Constructions

[0420] In some embodiments, fusion proteins CPI and SPP2 each comprise a biotinbinding portion of rhizavidin spanning amino acids 45 to 179 of the full-length protein, wherein the predicted signal sequences (amino acids 1-44) of rhizavidin were not incorporated. To optimize the expression level of CPI in E. coli, in some embodiments, the gene sequence that encodes rhizavidin polypeptide (e.g., in some embodiments, amino acids 45-179; SEQ ID NO: 2) was redesigned using E. co/z-preferred expression codons, synthesized and cloned into plasmid pET24a(+). This synthetic rhizavidin gene was designated Rhavi.

[0421] To construct the fusion proteins CPI and SPP2, in some embodiments, a DNA sequence encoding a flexible linker (e.g, in some embodiments, a flexible linker of GGGGSSS; SEQ ID NO: 28) was directly inserted into the 3’ end of the synthetic Rhavi gene to provide separation from Rhavi and promote proper folding of the subsequent fusion protein.

[0422] For CPI, the genes encoding desired portions of SP1500 and SP0785 protein (not including predicted signal sequences; nucleic acid sequences encoding the polypeptides of SEQ ID NO: 13 and SEQ ID NO: 11, respectively) were synthesized and inserted into the Rhavi expression vector just beyond the linker region. Three residual amino acids (AAA) from a Not I restriction site of pET24a(+) separate SP1500 and SP0785, and may be referred to as the second linker of CPI. Stop codons were included at the 3’-end of the CPI coding sequences. After cloning was complete, DNA sequencing of the pET-24a(+):CPl plasmid was performed to confirm the presence, orientation and sequence of the CPI DNA insert in the pET-24a(+) vector. A schematic of fusion protein CPI is shown in Figure 2.

[0423] For SPP2, a DNA sequence encoding an exemplary fusion protein SPP2 (Rhavi-GGGGSSS-PdT(G294P)-GGGGSSS-SP0435; e.g., SEQ ID NO: 32 or SEQ ID NO: 33) was cloned into the pET-24a(+) vector using PCR cloning. Stop codons were included at the 3 ’-end of the coding sequences. After cloning was complete, DNA sequencing of the pET-24a(+):SPP2 plasmid was performed to confirm the presence, orientation and sequence of the SPP2 DNA insert in the pET-24a(+) vector. A schematic of fusion protein SPP2 is shown in Figure 3. The map of plasmid pET24a(+):SPP2 is shown in Figure 4.

Establishment of Master Cell Banks (MCB)

[0424] To generate the Research Cell Banks (RCB), the pET-24a(+):CPl and pET24a(+):SPP2 plasmids were transformed into an E. coli expression strain. Transformed E. coli cells were plated in culture plates containing Kanamycin. A single colony from the plate was selected and used to inoculate into liquid medium in a shaker flask. The flasks were placed in an incubator shaker for overnight culture and grown to the desired OD. The bacterial culture was then mixed with glycerol solution. The mixed solution was aliquoted into vials to make RCB.

[0425] To generate the MCB, RCB was inoculated into liquid medium in shaker flasks and grown to the desired OD. The bacterial culture was then brought to 15% glycerol, mixed and aliquoted into vials. The MCB vials were then placed at -80°C (-70°C to -90°C) and selected vials were taken for quality assurance testing.

Manufacturing Process Overview

[0426] The CPI and SPP2 fusion proteins were expressed in E. coli. The expressed CPI and SPP2 fusion proteins were released from E. coli cells and purified in a series of chromatographic and filtration steps. The following section describes the 300 L culture scale CPI manufacturing process.

[0427] The process was initiated by thawing and inoculating cells from a MCB vial into cell medium. Initial cell expansion was performed in flask and then the bacterial culture was transferred to a 300 L fermenter. Bacteria were harvested by centrifugation. The recovered cell paste was resuspended in a lysis buffer and fluidized by a microfluidizer. Bulk CPI fusion protein in the fluidized cell lysate was purified by precipitation and chromatography steps. The final process stream was concentration and buffer exchange into 20 mM Tris, 150 mM NaCl at pH 8.0 ± 0.1 via ultrafiltration membranes. Lastly, the CPI fusion protein was 0.22 pm filtered immediately prior to bottling and stored at -80 °C. No raw materials contained animal-derived or human-derived components. Upstream Process

[0428] A flow chart of a representative upstream manufacturing process for fusion proteins CPI and SPP2 is provided in Figure 5, upper panel, and details for the individual steps follow.

[0429] The cell culture utilized chemically defined media and components depending on the manufacturing step. The media used were cell media, production media, feed 1 media and feed 2 media. These media were used in inoculum expansion, production culture, and feeding, respectively.

[0430] Step 1 - 1 : Thawing of Cells

Sufficient cell medium was prepared and transferred into an appropriate culture flask and then frozen MCB is thawed.

[0431] Step 1 - 2: Flask Expansion

Thawed MCB was inoculated and grown in a 2.5 L flask. Cell growth was monitored by a spectrophotometer at OD600.

[0432] Step 1 - 3: Production Culture

The production culture was run in fed-batch mode. To support product generation and to prolong the cell culture production period, feed 1 and feed 2 media were added after appropriate cell growth. Once the fermenter temperature reached the set point, isopropyl -D- 1-thiogalactopyranoside was added to induce fusion protein expression. After approximately 18 hours from induction, harvest was begun by centrifugation.

[0433] Step 1 - 4: Harvest

Bacterial culture was harvested by continuous flow centrifugation.

[0434] Step 1 - 5: Cell Lysis and Clarification

The recovered cell paste was resuspended in lysis buffer and fluidized by a microfluidizer. The fluidized cell lysate, including bulk fusion protein, was centrifuged and the supernatant was recovered.

[0435] Alternative upstream processes may be employed to manufacture the CPI and SPP2 fusion proteins.

Downstream Process [0436] After harvest and cell lysis, fusion proteins CPI and SPP2 were purified by several chromatography steps. A flow chart of a representative fusion protein purification process is provided in Figure 5, lower panel. Details for the individual steps follow.

[0437] Step 2 - 1 : Ammonium Sulfate Precipitation/Centrifugation and Filtration

Ammonium sulfate solution was added to the clarified lysate and adjusted to a final concentration of 0.8 M. After mixing at room temperature, the clarified lysate with ammonium sulfate was centrifuged and the supernatant was recovered. The recovered supernatant was filtered through 0.22 pm filter and collected in an appropriate container.

[0438] Step 2 - 2: Hydrophobic Interaction Chromatography

The purpose of this step is to remove process-related contaminants. Fusion protein bound to the resin was washed with high concentration ammonium sulfate buffer then eluted with elution buffer (the concentration of NaCl in the buffer was gradually reduced). A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed with each fraction to confirm the presence of fusion protein.

[0439] Step 2 - 3: Tangential Flow Filtration Concentration/Filtration

The elution pool was concentrated and then buffer exchanged with several-fold volumes of Tris-buffered saline using a 30 kDa filtration membrane. The filtrated tangential flow filtration (TFF) retentate was filtered by 0.22 pm filter.

[0440] Step 2 - 4: Anion Exchange Chromatography

The purpose of this step is to remove process-related contaminants. Fusion protein bound to the resin was washed with NaCl wash buffer then eluted with NaCl elution buffer (the concentration of NaCl in the buffer was gradually increased). SDS-PAGE analysis was performed with each fraction to confirm the presence of fusion protein.

[0441] Step 2 - 5: Type 1 Chromatography

The purpose of this step is to further remove process-related contaminants. Fusion protein bound to the resin was washed with phosphate wash buffer then eluted with phosphate elution buffer (the concentration of phosphate in the buffer was gradually increased). SDS-PAGE analysis was performed with each fraction to confirm the presence of fusion protein.

[0442] Step 2 - 6: TFF Formulation The Type 1 chromatography fraction pool was concentrated and then buffer exchanged with several-fold volumes of formulation buffer (20 mM Tris, 150 mM sodium chloride, pH 8.0 ± 0.1) using a 30 kDa filtration membrane. The formulated fusion protein was filtered through 0.22 pm filter.

[0443] Alternative downstream processes may be employed to manufacture the CPI and SPP2 fusion proteins.

Container Closure System

[0444] CPI and SPP2 fusion proteins were stored at -80°C (-70°C to -90°C) in sterile polyethylene terephthalate bottles.

Example 2: .S. pneumoniae Capsular Polysaccharides (PS) and Preparations of the Same

Preparation and Purification

[0445] Methods of culturing pneumococci are well known in the art (<?.g. Chase, 1967, Methods of Immunology and Immunochemistry 1:52). Methods of preparing pneumococcal capsular polysaccharides are also well known in the art (e.g. , European Patent No. EP0497524). Isolates of pneumococcal serotypes are available from the ATCC, the National Collection of Type Cultures operated by Public Health England, and other repositories.

[0446] S. pneumoniae is identified as non-motile, Gram-positive, lancet-shaped diplococci that are alpha-hemolytic on blood agar. Most but not all strains are encapsulated. Serotypes are differentiated on the basis of Neufeld Test (Quelling reaction) using specific antisera (e.g., U.S. Pat. No. 5,847,112), latex agglutination, or multilocus sequence typing.

[0447] A frozen vial representing each of the S. pneumoniae serotypes present in MAPS vaccine candidates of the present disclosure was thawed and used to generate a seed culture in appropriate pre- sterilized growth media. The seed culture was grown with temperature and pH control. The seed culture was transferred to a production fermenter that contained pre-sterilized growth media. The production culture was grown with temperature, pH and agitation rate control. The growth process was terminated with addition of an inactivating agent with a controlled temperature hold. [0448] The purification process was initiated by removal of cell debris using a combination of centrifugation and filtration. The material was filtered followed by solventbased fractionations to remove impurities and recover PS.

Exemplary polysaccharide structures

[0449] Representative polysaccharide (PS) structures are shown in Figure 6. All PS specifications in Table 1 were obtained from European Pharmacopoeia 9.0 Table 0966.-1, except for molecular size and serotype 6 A, which are based on the manufacturer’s certificate of analysis.

Table 1. Exemplary Polysaccharide (PS) Specifications

Values shown are percentage contents of components of monovalent bulk PS. t The different types are indicated using the Danish nomenclature.

$ Cross-linked agarose for chromatography R.

§ Cross-linked agarose for chromatography Rl .

Example 3: Preparation of MAPS Immunogenic Complexes

Overview

[0450] The MAPS platform provides various advantages including, e.g., 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 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 cross-reactivity to rhizavidin was observed in human serum samples obtained from subjects exposed to avidin [Helppolainen et al, 2007], 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]. The biotinylation of the PS and MAPS immunogenic complexing process in MAPS24 were optimized to consistently show no free biotin, thus reducing the potential for generating antibiotin antibodies.

[0451] MAPS vaccine candidates comprise genetically constructed fusion proteins of a biotin-binding moiety (e.g., rhizavidin, or a biotin-binding domain or biotin-binding fragment thereof), and a protein antigens of interest, which are then complexed with biotinylated PS of interest, leading to specific assembly into integrated macromolecular immunogenic complexes that, when processed by the immune system, result in the activation of protective B- and T-cell immune responses, as shown schematically in Figure 1.

[0452] MAPS34 is a pneumococcal vaccine candidates based on the proprietary MAPS platform. MAPS34 is a 34-valent MAPS vaccine candidate comprising 34 pneumococcal capsular polysaccharides of serotypes 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, 35B, and 38, individually biotinylated and complexed with either SPP2 or CPI fusion protein. MAPS vaccine candidates comprising at least 30 (e.g. , 30 [MAPS30], 31 [MAPS31], 32 [MAPS32], or 33 [MAPS33]) of the same 34 pneumococcal capsular polysaccharides as in MAPS34 are also contemplated. MAPS30+ refers to any one of MAPS30, MAPS31, MAPS32, MAPS33 or MAPS34 pneumococcal vaccine candidates.

[0453] In some embodiments, an exemplary SPP2 comprises a genetic fusion construct of truncated rhizavidin plus pneumococcal proteins pneumolysin (with a combination of four amino acid substitutions as described herein, denoted PdT(G294P)) and SP0435, joined by linkers, as shown in Figure 3. In some embodiments, an exemplary CPI comprises a genetic fusion construct of truncated rhizavidin plus pneumococcal proteins SP1500 and SP0785 joined by linkers, as shown in Figure 2. No available vaccines contain these unique combinations of PS and polypeptide antigens which are capable of eliciting an immune response against such a broad range of pneumococcal serotypes.

Assembly of MAPS Immunogenic Complexes

[0454] Drug substance (MAPS immunogenic complexes) comprises PS and CPI and SPP2 fusion protein. In some embodiments, since the PS are biotinylated and CPI and SPP2 fusion protein have a rhizavidin biotin-binding domain, they are linked by the high affinity biotin-rhizavidin interaction. MAPS immunogenic complexation was conducted individually for each PS serotype. [0455] In some embodiments, each MAPS immunogenic complex was made from 2 key intermediates: PS of each of up to 34 S. pneumoniae serotypes, and rhizavidin fusion proteins CPI or SPP2. PS of each serotype were activated, for example, by creation of a cyanate ester and then biotinylated. The biotinylated PS were mixed with CPI or SPP2 fusion protein to create MAPS immunogenic complexes of defined PS serotype and fusion protein, linked by the high affinity biotin-rhizavidin interaction. MAPS immunogenic complexes of defined PS serotype and fusion protein are referred to as a species.

[0456] MAPS immunogenic complexes of each species were formulated with 150 mM sodium chloride and surfactant buffer, then 0.2 pm filtered immediately prior to bottling and storing at 2°C to 8°C. Formulated MAPS immunogenic complexes of each species are referred to as MAPS drug substance.

[0457] No raw materials contained animal-derived or human-derived components.

[0458] A flow chart of a representative MAPS complexation process is provided in

Figure 7, and details for the individual steps follow.

[0459] Step 1: Clean-up of Polysaccharide

The purpose of this step is to remove process residuals. Dissolved PS was purified by filtration then exchanged with several-fold volumes of water for injection, followed by ultrafiltration to concentrate the PS. The cleaned-up PS was filtered by a 0.22 pm filter membrane.

[0460] Step 2: Biotinylation and Purification of Polysaccharide

The hydroxyl group on the PS was activated with a l-cyano-4-dimetylamino-pyridinium tetrafluoroborate (CDAP) to create a highly active cyanoester. The cyanoesters were reacted with amine-PEG3 -biotin and unreacted cyanoesters were capped with glycine. The biotinylated PS was buffer exchanged into 1 mM PBS in order to remove unreacted CDAP, amine-PEG3-biotin, glycine and residuals. After buffer exchange, the biotinylated PS was filtered with a 0.22 pm filter.

[0461] Step 3: MAPS immunogenic complexation

The biotinylated PS was mixed with CPI or SPP2 fusion protein in order to create the desired species of MAPS immunogenic complexes, linked by a high affinity biotin-rhizavidin interaction. The MAPS immunogenic complexes were purified to remove uncomplexed PS and protein by using a filtration membrane. After purification, the MAPS immunogenic complexes were filtered with a 0.22 pm filter and stored at 2°C to 8°C.

Specifications

[0462] Exemplary MAPS immunogenic complex/MAPS drug substance specifications are set forth in Table 2.

Table 2. Exemplary MAPS Drug Substance Specifications

FIO: for information only; PS: polysaccharide.

Example 4: MAPS34 Vaccine - Exemplary MAPS30+ Vaccine

Drug Product

[0463] MAPS34 is a 34-valent MAPS vaccine candidate comprising 34 pneumococcal capsular polysaccharides of serotypes 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, 35B, and 38, individually biotinylated and complexed with either SPP2 or CPI fusion protein. MAPS vaccine candidates comprising at least 30 (<?.g. , 30 [MAPS30], 31 [MAPS31], 32 [MAPS32], 33 [MAPS33]) of the same 34 pneumococcal capsular polysaccharides as in MAPS34 are also contemplated. MAPS30+ refers to any one of MAPS30, MAPS31, MAPS32, MAPS33, or MAPS34 pneumococcal vaccine candidates.

[0464] In a representative formulation, a MAPS34 vaccine candidate is formulated so that each 0.5-mL dose of MAPS34 drug product comprises 1, 2, 5, or 6 pg of each PS (for MAPS34, from each of 5. pneumoniae serotypes 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 35B, and 38, contributed by each species of MAPS immunogenic complex. MAPS34 is formulated for IM administration with aluminum phosphate adjuvant (Adju-Phos, from Croda Denmark, formerly Brenntag Biosector). In some embodiments, the total amount of aluminum per dose is 0.625 mg, which is below the FDA/WHO maximum recommended dose of 0.85 mg to 1.25 mg. In some embodiments, the total amount of aluminum per dose is less than 0.625 mg. In some embodiments, the total amount of aluminum per dose is between 0.325 mg and 0.175 mg. In some embodiments, the total amount of aluminum per dose is 0.25 mg. In some embodiments, the total amount of aluminum per dose is at least 0.125 mg. Figure 8 shows a representative scheme for MAPS34 drug product manufacturing.

Example 5: Exemplary Pharmaceutical Compositions with a Reduced Amount of an Aluminum-Based Adjuvant and Their Immunogenicity

[0465] The goal of this study was to further define the preferred combinations of S. pneumoniae polysaccharides (serotypes) with antigenic fusion proteins CPI and SPP2, in order to optimize the immune response profile of 34- valent MAPS vaccine candidates. The vaccine candidates used in this study comprised different combinations of 34 .S', pneumoniae polysaccharides and fusion protein CPI only, or both fusion proteins SPP2 and CPI, as shown in Figure 9 and Table 3, below. Immunization group T comprised the same combination of polysaccharides and fusion proteins SPP2 and CPI as immunization group S, with aluminum (as aluminum phosphate) at a reduced concentration of 0.05 mg/mL (0.025 mg per vaccine dose). For all rabbit immunization groups, MAPS complexes and aluminum (as aluminum phosphate) were present at l/10^th the human dose.

Production of Rabbit Hyperimmune Sera [0466] Hyperimmune sera were obtained using 4 or more groups of New Zealand White rabbits (Cocalico Biologicals), each comprising up to 20 rabbits. On Day 0, groups of rabbits were immunized intramuscularly according to Table 3 below. A second immunization was administered on Day 14. Serum was collected on Day 0 prior to first immunization (P0 serum), on Day 14 prior to second immunization (post-first immunization production bleed; Pl serum), and on Day 28 (post-second immunization production bleed; P2 serum).

Table 3. Immunization Groups for Production of Rabbit Hyperimmune Sera

IgG Antibody Responses to S. Pneumoniae Polysaccharides

[0467] IgG antibody levels specific to the 24 polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 22F, 23F, and 33F (also included in Applicant’s previously described vaccine, see WO 2020/056202), and 10 additional polysaccharide serotypes 6C, 7C, 15A, 16F, 23 A, 23B, 24F, 31, 35B, and 38 were evaluated in P2 sera collected from immunized rabbits (above). An electro- chemiluminescent-based Meso Scale Discovery immunoassay (MSD) was performed using a reference standard. Briefly, 3-fold serial dilutions were created starting with a 1:200 dilution of the reference standard in 1% Casein, 5 g/mL CWPS, and 0.05% Tween-20. The control and experimental rabbit serum samples were diluted 1000-fold and 5000-fold with 1% Casein, 5 pg/mL CWPS, and 0.05% Tween-20, respectively. Plates were washed with IX PBS-T prior to adding the reference standard as well as the control and experimental rabbit sera. After incubating at room temperature for one hour, plates were washed with IX PBS-T and secondary anti-rabbit SULFO-conjugated antibody was added. The plates were washed with IX PBS-T; IX Read Buffer was added and the plates were read using an MSD Meso QuickPlex SQ 120 Model No. 1300.

[0468] In a first analysis, antibody levels were graphed as arbitrary units (a.u.) for the top figure and in ng/mL for the bottom figure (A, B, C, T and S). Figure 10 shows representative IgG levels against 34 capsular polysaccharide serotypes in P2 rabbit sera from each of immunization groups A, B, C, S and T. The top panel displays results for the first 24 serotypes. The bottom panel displays results for the additional 10 serotypes.

[0469] In another analysis, IgG levels measured in sera from each immunization group were compared pair- wise to a baseline. 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 the baseline of MAPS-34 (all-on-CPl) [immunization group B]. 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]. For each comparison, IgG levels against S. pneumoniae polysaccharides were expressed as geometric mean titer (GMT) ratio at 95% confidence intervals, relative to the baseline represented as a dotted line at GMT ratio of 1. If the point estimate of the GMT ratio > 1 , then anti-polysaccharide antibody levels for a given immunization group were higher than the baseline. If the point estimate of the GMT ratio < 1 , then anti-polysaccharide antibody levels for a given immunization group were lower than the baseline. In Figure 11, 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.

[0470] These results show that the MAPS-34 (24-CP1/10-SPP2) [immunization group C], MAPS-34 (selected 24/10) [immunization group S], and MAPS-34 (selected 24/10, lower dose of aluminum) [immunization group T] configurations of capsular polysaccharides with fusion proteins SPP2 and CPI trended towards overall greater IgG responses to polysaccharides as compared to MAPS-34 (all-on-CPl) [immunization group B], IgG responses to polysaccharides were overall highest in the MAPS-34 (selected 24/10, lower dose of aluminum) [immunization group T] configuration.

IgG Responses to Proteins

[0471] To measure titers of rabbit IgG antibody against the 5. pneumoniae portions of the fusion proteins CPI and SPP2, an Enzyme-Linked Immunosorbent Assays (ELISA) specific for each fusion protein was performed. For CPI, IgG response was assessed against the SP1500-SP0785 portion of the fusion protein. For SPP2, IgG response was assessed against the PdT(G294P) portion of the fusion protein.

[0472] MaxiSorp 96-well plates (ThermoFisher Scientific, Waltham, MA, USA) were coated with 100 pl per well of 2.0 pg/ml SP1500-SP0785, or PdT(G294P) in IX Dulbecco’s phosphate-buffered saline (DPBS) overnight at room temperature. Plates were washed with IX DPBS with 0.05 % Tween 20 (DPBS-T) and then blocked with 200 pl per well of 1 % BSA in DPBS-T for 1 hour. Plates were washed with IX DPBS-T prior to the addition of rabbit sera for testing diluted in DPBS-T. Primary antisera dilutions were incubated for 1 hour at room temperature followed by washing with DPBS-T. Goat anti-rabbit IgG (Fc)-HRP (Jackson ImmunoResearch, West Grove, PA, USA) was diluted 1: 100,000 and 100 pl was added to each well, followed by a 1-hour incubation at room temperature. Plates were washed with DPBS-T; 100 pl of tetramethylbenzidine (TMB peroxidase substrate - KPL) was added to each well, incubated for 30 minutes, and stopped with 100 pl of 1 N HC1. Absorbance was read on a SpectraMax i3x at OD45o- Absorbance values were analyzed using SoftMax Pro and Microsoft Excel utilizing a 4-parameter logistic curve fit for a standard with known concentration. Geometric mean titers (GMTs) were calculated by transforming individual antibody titers to their logw concentrations, taking the means of the transformed concentrations from each animal group at each time point, and subsequently transforming those means to GMTs by taking their anti-logio. The asymmetric 95 % confidence intervals (CI) were calculated by the GraphPad Prism Software (version 7, La Jolla, CA, USA) together with the GMTs.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] With respect to SP 1500-SP0785, these results show that the MAPS-34 (24-

CP1/10-SPP2) [immunization group C] and MAPS-34 (selected 24/10) [immunization group S] configurations of capsular polysaccharides with fusion proteins SPP2 and CPI produced an approximately 2-fold greater IgG response than MAPS -23 [immunization group A] or MAPS-34 (all-on-CPl) [immunization group B]. The MAPS-34 (selected 24/10, lower dose of aluminum) [immunization group T] configuration resulted in lower IgG response.

[0477] With respect to PdT(G294P), the MAPS-34 (24-CP1/10-SPP2) [immunization group C] and MAPS-34 (selected 24/10, lower dose of aluminum) [immunization group T] configurations produced an approximately 2-fold greater IgG response than MAPS-34 (selected 24/10) [immunization group S]. As expected, the MAPS-23 [immunization group A] and MAPS-34 (all-on-CPl) [immunization group B] configurations, comprising only the CPI fusion protein, produced only background responses. The IgG responses to SP1500- SP0785 and PdT(G294P) were not negatively correlated, suggesting no interference.

Neutralizing Antibody Responses

[0478] Pneumolysin neutralization assays were performed to determine whether antibodies against fusion protein SPP2 (specifically, the PdT(G294P) portion of SPP2) are able to neutralize the hemolytic activity of native pneumolysin (red blood cell lysis). Briefly, 50 pl of a 200 ng/ml pneumolysin solution in lx PBS, 0.1% bovine serum albumin and 10 mM dithiothreitol (DTT) was incubated with 50 l of serum serial dilutions from rabbits in a V-bottom 96-microwell plate for 30 min at 37°C with 5% CO2 and shaking at 350 rpm. Following the initial incubation period, 50 pl of 2% rabbit red blood cells was added and incubated for an additional 30 min using the same conditions. After centrifugation at 1000 x g for 5 min to pellet intact red blood cells, the supernatants were harvested and the absorbance at 545 nm was measured to quantify the extent of hemolysis. Absorbance was read on a SpectraMax i3x at OD545. Absorbance values were analyzed using SoftMax Pro and Microsoft Excel utilizing a 4-parameter logistic curve fit to determine the serum dilution at which 50% of the hemolytic activity of pneumolysin was inhibited (IC50). For samples that do not achieve 50% neutralization, the lowest dilution tested was recorded. Geometric mean titers (GMTs) were calculated by transforming individual IC50s to their logic concentrations, taking the means of the transformed concentrations from each animal group at each time point, and subsequently transforming those means to GMTs by taking their anti-log 10. The asymmetric 95% confidence intervals (CI) were calculated by the GraphPad Prism Software (version 7, La Jolla, CA, USA) together with the GMTs.

[0479] 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.

[0480] 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.

[0481] These results show that the MAPS-34 (selected 24/10) [immunization group S] configuration elicited lower pneumolysin-neutralizing titers than MAPS-34 (24-CP1/10- SPP2) [immunization group C] or MAPS-34 (selected 24/10, lower dose of aluminum) [immunization group T]. IgG response against PdT(G294P) was strongly correlated with pneumolysin-neutralizing titers above ~30ug/mL.

Example 6: Aluminum-Based Adjuvant Formulation Immunogenicity Assessment

[0482] This study assessed the effect of aluminum concentration on immunogenicity of MAPS vaccine candidates. The vaccine candidates used in this study included different combinations of 5. pneumoniae polysaccharides with fusion protein CPI only, or both fusion proteins SPP2 and CPI, as shown in Figure 18 and Table 4 below. Formulations included 1.25 mg/mL aluminum (High), 0.5 mg/mL aluminum (Medium), or 0.25 mg/mL (Low). Aluminum (as aluminum phosphate) formulation concentrations were tested across three concentrations: 0.025 mg/mL (0.0125 mg per vaccine dose, “low”), 0.05 mg/mL (0.025 mg per vaccine dose; “medium”), and 0.125 mg/mL (0.0625 mg per vaccine dose; “high”). For all rabbit immunization groups, MAPS complexes and aluminum (as aluminum phosphate) were present at 1 /10^th the human dose.

Table 4. Immunization Groups for Production of Rabbit Hyperimmune Sera

Production of Rabbit Hyperimmune Sera

[0483] Hyperimmune sera were obtained using New Zealand White male rabbits (Cocalico Biologicals) from three immunization vaccine groups, where three separate aluminum formulation adjuvant groups (per vaccine group) were tested comprising up to 15 rabbits each. On Day 0, alum formulation adjuvant rabbit groups were immunized intramuscularly according to Table 4. A second immunization was administered on Day 14. Serum was collected on Day 0 prior to first immunization (P0 serum), on Day 14 prior to second immunization (post- first immunization production bleed; Pl serum), and on Day 28 (post-second immunization production bleed; P2 serum).

IgG Antibody Responses to S. Pneumoniae Polysaccharides

[0484] IgG antibody responses to S. pneumoniae polysaccharides were assessed as described in Example 5. [0485] Figure 19A shows representative IgG levels against 24 capsular polysaccharide serotypes in P2 rabbit sera for MAPS -24 (All on CPI) from each of the aluminum groups (Group C: Low, Group B: Med, and Group A: High) (antibody levels were graphed as arbitrary units (a.u.)). In another analysis (depicted in Figure 19B), IgG levels measured in sera from Group B (Medium) and Group C (Low) were compared pair- wise to baseline IgG levels measured in sera for Group A (High). IgG antibody levels were specific to 24 polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20B, 22F, 23F, and 33F. These results show that Group C (Low) trended towards overall greater IgG responses to polysaccharides as compared to Group A (High).

[0486] Figure 21 A shows representative IgG levels against the 24 polysaccharide serotypes listed above, in addition to 10 additional polysaccharide serotypes 6C, 7C, 15 A, 16F, 23A, 23B, 24F, 31, 35B, and 38 for MAPS-34 (All on CPI) from each of the aluminum groups (Group C: Low, Group B: Med, and Group A: High) (antibody levels were graphed as arbitrary units (a.u.)). In another analysis (depicted in Figure 2 IB), IgG levels measured in sera from Group B (Medium) and Group C (Low) were compared pair- wise to baseline IgG levels measured in sera for Group A (High). These results show that Group B (Med) trended towards overall greater IgG responses to polysaccharides as compared to Group A (High).

[0487] Figure 23A shows representative IgG levels against the 34 capsular polysaccharide serotypes included in MAPS-34 (24V-CP1 / 10V-SPP2) from each of the aluminum groups (Group C: Low, Group B: Med, and Group A: High) (antibody levels were graphed as arbitrary units (a.u.)). In another analysis (depicted in Figure 23B), IgG levels measured in sera from Group B (Medium) and Group C (Low) were compared pair- wise to baseline IgG levels measured in sera for Group A (High). These results show that Group C (Low) trended towards overall greater IgG responses to polysaccharides as compared to Group A (High). Notably, for MAPS-34 (All on CPI), IgG responses to certain polysaccharide subtypes (e.g., 31, 35B, and 38) trended lower in Group C (Low) relative to Group A (High). However, for MAPS-34 (24V-CP1 / 10V-SPP2), IgG responses to the same polysaccharide subtypes (e.g., 31, 35B, and 38) trended greater in Group C (Low) relative to Group A (High).

[0488] For each comparison in Figures 19B, 21B, and 23B, IgG levels against S. pneumoniae polysaccharides were expressed as geometric mean titer (GMT) ratios at 95% confidence intervals, relative to the baseline Group A (High Alum) represented as a dotted line at GMT ratio of 1. If the point estimate of the GMT ratio > 1, then anti- polysaccharide antibody levels for a given immunization group were higher than the baseline Group A (High Alum). If the point estimate of the GMT ratio < 1, then anti-polysaccharide antibody levels for a given immunization group were lower than the baseline Group A (High Alum).

IgG Antibody Responses to Proteins

[0489] Titers of rabbit IgG antibody against the S. pneumoniae portions of the fusion proteins CPI and SPP2 were assessed as described in Example 5.

[0490] Figure 20 shows representative IgG levels against the A pneumoniae SP1500- 0785 portion of CPI fusion protein in Pl and P2 rabbit sera for MAPS-24 (All on CPI) from Group A (High), Group B (Medium), and Group C (Low). Figure 22 shows representative IgG levels against the S'. pneumoniae SP 1500-0785 portion of CPI fusion protein in Pl and P2 rabbit sera for MAPS-34 (All on CPI) from Group A (High), Group B (Medium), and Group C (Low). Figure 24 A shows representative IgG levels against the S. pneumoniae SP1500-0785 portion of CPI fusion protein in Pl and P2 rabbit sera for MAPS-34 (24V-CP1 / 10V-SPP2) from Group A (High), Group B (Medium), and Group C (Low). Figure 24B shows representative IgG levels against the S. pneumoniae PdT (G294P) portion of SPP2 fusion protein in Pl and P2 rabbit sera for MAPS-34 (24V-CP1 / 10V-SPP2) from Group A (High), Group B (Medium), and Group C (Low). Each dot on the graphs represents one rabbit. Results are expressed in ug/mL with 95% confidence intervals on the graphs and tabulated as geometric means ( g/mL) below the graphs.

[0491] With respect to SP1500-SP0785, these results show that for MAPS-24 (All on CPI), Group B (Medium) and Group C (Low) resulted in lower IgG response relative to Group A (High). For MAPS-34 (All on CPI), Group A (High) and Group C (Low) resulted in lower IgG response relative to Group B (Medium).

[0492] With respect to SP1500-SP0785, Group B (Medium) resulted in lower IgG response relative to Group A (High) and Group C (Low). With respect to PdT (G294P), these results show that for MAPS-34 (24V-CP1 / 10V-SPP2), for P2 sera, Group B (Medium) and Group C (Low) resulted in lower IgG response relative to Group A (High). List of Sequences

[0494] SEQ ID NO: 1, Rhizavidin protein, full-length [aa 1-179]:

MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALAFDASNFKDFSSIAS ASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVG WNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPT TENKSLLKD

[0495] SEQ ID NO: 2, truncated rhizavidin protein [aa 45-179], denoted Rhavi:

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLT GRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAI EQGQDTFQYVPTTENKSLLKD

[0496] SEQ ID NO: 3, truncated rhizavidin protein [aa 45-179], denoted Rhavi, includes leading methionine:

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYP LTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGP AIEQGQDTFQYVPTTENKSLLKD

[0497] SEQ ID NO: 4, Streptococcus pneumoniae Pneumolysin protein:

MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNT SDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQV EDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFE KTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRG ISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNT EVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVA TFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAW

DRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTIS IWGTTLYPQVEDKVEND

[0498] SEQ ID NO: 5, Streptococcus pneumoniae Pneumolysin protein with mutations D385N, C428G, and W433F, denoted PdT:

MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNT SDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQV EDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFE KTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRG

ISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNT

EVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVA

TFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAW

DRNGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTIS IWGTTLYPQVEDKVEND

[0499] SEQ ID NO: 6, Streptococcus pneumoniae Pneumolysin protein with mutations G294P, D385N, C428G, and W433F, denoted PdT(G294P):

MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNT

SDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQV

EDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFE

KTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRG

ISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNT

EVKAVILGPDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVA

TFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAW

DRNGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTIS IWGTTLYPQVEDKVEND

[0500] SEQ ID NO: 7, Streptococcus pneumoniae Pneumolysin PdT(G294P) [aa 2-

470] protein:

ANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSD

ISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVED

PSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKT

GNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGIS

AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTE

VKAVILGPDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATF

QNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAWDR NGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIW GTTLYPQVEDKVEND

[0501] SEQ ID NO: 8, Streptococcus pneumoniae SP0435 protein, full length, TIGR4 strain (GenBank: ABJ54475.1):

MIEASKLKAGMTFETADGKLIRVLEASHHKPGKGNTIMRMKLRDVRTGSTFDTSYR

PEEKFEQAIIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKI QFYGTEVIGVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQ

KLVINTAEGTYVSRA

[0502] SEQ ID NO: 9, Streptococcus pneumoniae SP0435 [aa 62-185] protein,

TIGR4 strain:

EQAIIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYG TEVIGVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVIN TAEGTYVSR

[0503] SEQ ID NO: 10, SP0785 protein, full-length [aa 1-399], TIGR4 strain: (Note: One T394A mismatch with SP0785 NCBI Sequences ABJ54007.1 and YP816180)

MKKKNGKAKKWQLYAAIGAASVVVLGAGGILLFRQPSQTALKDEPTHLVVAKEGS

VASSVLLSGTVTAKNEQYVYFDASKGDLDEILVSVGDKVSEGQALVKYSSSEAQAA

YDSASRAVARADRHINELNQARNEAASAPAPQLPAPVGGEDATVQSPTPVAGNSVA

SIDAQLGDARDARADAAAQLSKAQSQLDATTVLSTLEGTVVEVNSNVSKSPTGASQ

VMVHIVSNENLQVKGELSEYNLANLSVGQEVSFTSKVYPDKKWTGKLSYISDYPKN

NGEAASPAAGNNTGSKYPYTIDVTGEVGDLKQGFSVNIEVKSKTKAILVPVSSLVMD DSKNYVWIVDEQQKAKKVEVSLGNADAENQEITSGLTNGAKVISNPTSSLEEGKEV KADEATN

[0504] SEQ ID NO: 11, SP0785 protein lacking signal sequence [aa 33-399]: (Note: One T394A mismatch with SP0785 NCBI Sequences ABJ54007.1 and YP816180)

FRQPSQTALKDEPTHLVVAKEGSVASSVLLSGTVTAKNEQYVYFDASKGDLDEILVS

VGDKVSEGQALVKYSSSEAQAAYDSASRAVARADRHINELNQARNEAASAPAPQLP

APVGGEDATVQSPTPVAGNSVASIDAQLGDARDARADAAAQLSKAQSQLDATTVLS

TLEGTVVEVNSNVSKSPTGASQVMVHIVSNENLQVKGELSEYNLANLSVGQEVSFTS

KVYPDKKWTGKLSYISDYPKNNGEAASPAAGNNTGSKYPYTIDVTGEVGDLKQGFS VNIEVKSKTKAILVPVSSLVMDDSKNYVWIVDEQQKAKKVEVSLGNADAENQEITS GLTNGAKVISNPTS SLEEGKEVKADEATN

[0505] SEQ ID NO: 12, SP1500 protein, full-length [aa 1-278], TIGR4 strain:

MKKWMLVLVSLMTALFLVACGKNSSETSGDNWSKYQSNKSITIGFDSTFVPMGFAQ

KDGSYAGFDIDLATAVFEKYGITVNWQPIDWDLKEAELTKGTIDLIWNGYSATDERR EKVAFSNSYMKNEQVLVTKKSSGITTAKDMTGKTLGAQAGSSGYADFEANPEILKNI VANKEANQYQTFNEALIDLKNDRIDGLLIDRVYANYYLEAEGVLNDYNVFTVGLET

EAFAVGARKEDTNLVKKINEAFSSLYKDGKFQEISQKWFGEDVATKEVKEGQ

[0506] SEQ ID NO: 13, SP1500 [aa 27-278]:

TSGDNWSKYQSNKSITIGFDSTFVPMGFAQKDGSYAGFDIDLATAVFEKYGITVNWQ

PIDWDLKEAELTKGTIDLIWNGYSATDERREKVAFSNSYMKNEQVLVTKKSSGITTA

KDMTGKTLGAQAGSSGYADFEANPEILKNIVANKEANQYQTFNEALIDLKNDRIDGL

LIDRVYANYYLEAEGVLNDYNVFTVGLETEAFAVGARKEDTNLVKKINEAFSSLYK

DGKFQEISQKWFGEDVATKEVKEGQ

[0507] SEQ ID NO: 14, Ply gene encoding Ply protein, full-length [aa 1-470]

ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAG

AAACTCTTGACCCATCAGGGAGAAAGTATTGAAAATCGTTTCATCAAAGAGGGT

AATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCG

ACAAATACAAGTGATATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTG

GAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACTCTTCTTGC

GGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGC

GATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTCGCGGAGCGGTA

AACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAG

CTAGAATGCAGCATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCA

AGTTTGGTTCTGACTTTGAAAAGATAGGGAATTCTCTTGATATTGATTTTAACTCT

GTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATA

CAGTCAGCGTAGATGCTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAAC

GGTAGAGGATTTAAGGCAGAGAGGAATTTCTGCAGAGCGTCCTTTGGTCTATATT

TCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGA

GTGATGAAGTAGAGGCTGCTTTTGAATCTTTGATAAAAGGAGTAGCTCCTCAGAC

AGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGGG

CGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGA

CTTGATTCAAGAAGGCAGTCGCTTTACAGCCGATCATCCAGGCTTGCCGATTTCC

TATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAG

ACTATGTTGAGACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCA

TAGTGGTGCCTATGTTGCTCAATATTATATTACTTGGGATGAATTATCCTATGATC

ATCAAGGCAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATT

TGACGGCTCACTTTACCACTAGTATTCCTTTAAAAGGGAATGTTCGCAATCTCTCT GTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTATG

AAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAA CTCTCTATCCTCAGGTAGAGGATAAGGTAGAAAATGATTAG

[0508] SEQ ID NO: 15, codon-optimized nucleotide sequence encoding PdT(G294P) [aa 1 -470]:

ATGGCGAACAAGGCGGTGAACGATTTTATCCTGGCGATGAACTATGACAAGAAG

AAACTGCTGACCCACCAAGGCGAGAGCATTGAGAACCGTTTCATTAAAGAAGGC

AACCAGCTGCCGGACGAGTTTGTGGTTATCGAGCGTAAGAAACGTAGCCTGAGC

ACCAACACCAGCGACATTAGCGTGACCGCGACCAACGATAGCCGTCTGTACCCG

GGTGCGCTGCTGGTTGTGGATGAAACCCTGCTGGAAAACAACCCGACCCTGCTG

GCGGTGGACCGTGCGCCGATGACCTATAGCATCGATCTGCCGGGTCTGGCGAGC

AGCGACAGCTTCCTGCAAGTTGAGGATCCGAGCAACAGCAGCGTGCGTGGTGCG

GTTAACGACCTGCTGGCGAAGTGGCACCAGGATTACGGCCAAGTGAACAACGTT

CCGGCGCGTATGCAGTATGAAAAAATCACCGCGCACAGCATGGAGCAACTGAAG

GTTAAATTCGGTAGCGACTTTGAAAAGACCGGCAACAGCCTGGACATTGATTTCA

ACAGCGTGCACAGCGGCGAGAAGCAGATCCAAATCGTTAACTTCAAGCAGATCT ACTACACCGTGAGCGTTGACGCGGTGAAGAACCCGGGTGACGTTTTCCAGGATA

CCGTGACCGTTGAAGATCTGAAACAACGTGGCATTAGCGCGGAGCGTCCGCTGG

TGTACATCAGCAGCGTTGCGTACGGTCGTCAAGTGTATCTGAAGCTGGAAACCAC

CAGCAAAAGCGATGAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAGGGCGTGAA

AGTTGCGCCGCAGACCGAATGGAAGCAAATTCTGGACAACACCGAGGTGAAAGC

GGTTATTCTGGGCCCGGATCCGAGCAGCGGCGCGCGTGTGGTTACCGGTAAAGT

GGACATGGTTGAGGATCTGATTCAGGAAGGTAGCCGTTTTACCGCGGACCACCC

GGGCCTGCCGATCAGCTACACCACCAGCTTCCTGCGTGACAACGTGGTTGCGACC

TTTCAAAACAGCACCGATTACGTGGAAACCAAGGTTACCGCGTATCGTAACGGT GACCTGCTGCTGGACCACAGCGGTGCGTACGTGGCGCAGTACTATATCACCTGG GATGAACTGAGCTATAACCACCAGGGTAAAGAGGTGCTGACCCCGAAAGCGTGG

GACCGTAACGGCCAGGATCTGACCGCGCACTTCACCACCAGCATTCCGCTGAAG

GGCAACGTGCGTAACCTGAGCGTTAAAATCCGTGAGGGTACCGGCCTGGCGTTT

GAATGGTGGCGTACCGTGTACGAGAAGACCGACCTGCCGCTGGTTCGTAAACGT

ACCATCAGCATTTGGGGTACCACCCTGTATCCGCAGGTGGAGGACAAAGTTGAA AATGAT [0509] SEQ ID NO: 16, codon-optimized nucleotide sequence encoding PdT(G294P) [aa 2-470]:

GCGAACAAGGCGGTGAACGATTTTATCCTGGCGATGAACTATGACAAGAAGAAA

CTGCTGACCCACCAAGGCGAGAGCATTGAGAACCGTTTCATTAAAGAAGGCAAC

CAGCTGCCGGACGAGTTTGTGGTTATCGAGCGTAAGAAACGTAGCCTGAGCACC

AACACCAGCGACATTAGCGTGACCGCGACCAACGATAGCCGTCTGTACCCGGGT

GCGCTGCTGGTTGTGGATGAAACCCTGCTGGAAAACAACCCGACCCTGCTGGCG

GTGGACCGTGCGCCGATGACCTATAGCATCGATCTGCCGGGTCTGGCGAGCAGC

GACAGCTTCCTGCAAGTTGAGGATCCGAGCAACAGCAGCGTGCGTGGTGCGGTT

AACGACCTGCTGGCGAAGTGGCACCAGGATTACGGCCAAGTGAACAACGTTCCG

GCGCGTATGCAGTATGAAAAAATCACCGCGCACAGCATGGAGCAACTGAAGGTT

AAATTCGGTAGCGACTTTGAAAAGACCGGCAACAGCCTGGACATTGATTTCAAC

AGCGTGCACAGCGGCGAGAAGCAGATCCAAATCGTTAACTTCAAGCAGATCTAC

TACACCGTGAGCGTTGACGCGGTGAAGAACCCGGGTGACGTTTTCCAGGATACC

GTGACCGTTGAAGATCTGAAACAACGTGGCATTAGCGCGGAGCGTCCGCTGGTG

TACATCAGCAGCGTTGCGTACGGTCGTCAAGTGTATCTGAAGCTGGAAACCACC

AGCAAAAGCGATGAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAGGGCGTGAAA

GTTGCGCCGCAGACCGAATGGAAGCAAATTCTGGACAACACCGAGGTGAAAGCG

GTTATTCTGGGCCCGGATCCGAGCAGCGGCGCGCGTGTGGTTACCGGTAAAGTG

GACATGGTTGAGGATCTGATTCAGGAAGGTAGCCGTTTTACCGCGGACCACCCG

GGCCTGCCGATCAGCTACACCACCAGCTTCCTGCGTGACAACGTGGTTGCGACCT

TTCAAAACAGCACCGATTACGTGGAAACCAAGGTTACCGCGTATCGTAACGGTG

ACCTGCTGCTGGACCACAGCGGTGCGTACGTGGCGCAGTACTATATCACCTGGG

ATGAACTGAGCTATAACCACCAGGGTAAAGAGGTGCTGACCCCGAAAGCGTGGG

ACCGTAACGGCCAGGATCTGACCGCGCACTTCACCACCAGCATTCCGCTGAAGG

GCAACGTGCGTAACCTGAGCGTTAAAATCCGTGAGGGTACCGGCCTGGCGTTTG

AATGGTGGCGTACCGTGTACGAGAAGACCGACCTGCCGCTGGTTCGTAAACGTA

CCATCAGCATTTGGGGTACCACCCTGTATCCGCAGGTGGAGGACAAAGTTGAAA ATGAT

[0510] SEQ ID NO: 17, SP0435 gene encoding SP0435 protein, full-length [aa 1-

186], TIGR4 strain: ATGATTGAAGCAAGTAAATTAAAAGCTGGTATGACCTTTGAAACAGCTGACGGC

AAATTGATTCGCGTTTTGGAAGCTAGTCACCACAAACCAGGTAAAGGAAACACG

ATCATGCGTATGAAATTGCGTGATGTCCGTACTGGTTCTACATTTGACACAAGCT

ACCGTCCAGAGGAAAAATTTGAACAAGCTATTATCGAGACTGTCCCAGCTCAAT

ACTTGTACAAAATGGATGACACAGCATACTTCATGAATACAGAAACTTATGACC

AATACGAAATCCCTGTAGTCAATGTTGAAAACGAATTGCTTTACATCCTTGAAAA

CTCTGATGTGAAAATCCAATTCTACGGAACTGAAGTGATCGGTGTCACCGTTCCT

ACTACTGTTGAGTTGACAGTTGCTGAAACTCAACCATCTATCAAAGGTGCTACTG

TTACAGGTTCTGGTAAACCAGCAACGATGGAAACTGGACTTGTCGTAAACGTTCC

AGACTTCATCGAAGCAGGACAAAAACTCGTTATCAACACTGCAGAAGGAACTTA CGTTTCTCGTGCC

[0511] SEQ ID NO: 18, SP0785 gene encoding SP0785 protein, full-length [aa 1-

399], TIGR4 strain:

ATGAAGAAAAAGAATGGTAAAGCTAAAAAGTGGCAACTGTATGCAGCAATCGGT

GCTGCGAGTGTAGTTGTATTGGGTGCTGGGGGGATTTTACTCTTTAGACAACCTT

CTCAGACTGCTCTAAAAGATGAGCCTACTCATCTTGTTGTTGCCAAGGAAGGAAG

CGTGGCCTCCTCTGTTTTATTGTCAGGGACAGTAACAGCAAAAAATGAACAATAT

GTTTATTTTGATGCTAGTAAGGGTGATTTAGATGAAATCCTTGTTTCTGTGGGCGA

TAAGGTCAGCGAAGGGCAGGCTTTAGTCAAGTACAGTAGTTCAGAAGCGCAGGC

GGCCTATGATTCAGCTAGTCGAGCAGTAGCTAGGGCAGATCGTCATATCAATGA

ACTCAATCAAGCACGAAATGAAGCCGCTTCAGCTCCGGCTCCACAGTTACCAGC

GCCAGTAGGAGGAGAAGATGCAACGGTGCAAAGCCCAACTCCAGTGGCTGGAA

ATTCTGTTGCTTCTATTGACGCTCAATTGGGTGATGCCCGTGATGCGCGTGCAGA

TGCTGCGGCGCAATTAAGCAAGGCTCAAAGTCAATTGGATGCAACAACTGTTCTC

AGTACCCTAGAGGGAACTGTGGTCGAAGTCAATAGCAATGTTTCTAAATCTCCAA

CAGGGGCGAGTCAAGTTATGGTTCATATTGTCAGCAATGAAAATTTACAAGTCAA

GGGAGAATTGTCTGAGTACAATCTAGCCAACCTTTCTGTAGGTCAAGAAGTAAG

CTTTACTTCTAAAGTGTATCCTGATAAAAAATGGACTGGGAAATTAAGCTATATT

TCTGACTATCCTAAAAACAATGGTGAAGCAGCTAGTCCAGCAGCCGGGAATAAT

ACAGGTTCTAAATACCCTTATACTATTGATGTGACAGGCGAGGTTGGTGATTTGA

AACAAGGTTTTTCTGTCAACATTGAGGTTAAAAGCAAAACTAAGGCTATTCTTGT

TCCTGTTAGCAGTCTAGTAATGGATGATAGTAAAAATTATGTCTGGATTGTGGAT GAACAACAAAAGGCTAAAAAAGTTGAGGTTTCATTGGGAAATGCTGACGCAGAA AATCAAGAAATCACTTCTGGTTTAACGAACGGTGCTAAGGTCATCAGTAATCCAA CATCTTCCTTGGAAGAAGGAAAAGAGGTGAAGGCTGATGAAGCAACTAAT

[0512] SEQ ID NO: 19, SP1500 gene encoding SP1500 protein, full-length [aa 1-

278], TIGR4 strain:

ATGAAAAAATGGATGCTTGTATTAGTCAGTCTGATGACTGCTTTGTTCTTAGTAG

CTTGTGGGAAAAATTCTAGCGAAACTAGTGGAGATAATTGGTCAAAGTACCAGT

CTAACAAGTCTATTACTATTGGATTTGATAGTACTTTTGTTCCAATGGGATTTGCT

CAGAAAGATGGTTCTTATGCAGGATTTGATATTGATTTAGCTACAGCTGTTTTTG

AAAAATACGGAATCACGGTAAATTGGCAACCGATTGATTGGGATTTGAAAGAAG

CTGAATTGACAAAAGGAACGATTGATCTGATTTGGAATGGCTATTCCGCTACAGA

CGAACGCCGTGAAAAGGTGGCTTTCAGTAACTCATATATGAAGAATGAGCAGGT

ATTGGTTACGAAGAAATCATCTGGTATCACGACTGCAAAGGATATGACTGGAAA

GACATTAGGAGCTCAAGCTGGTTCATCTGGTTATGCGGACTTTGAAGCAAATCCA GAAATTTTGAAGAATATTGTCGCTAATAAGGAAGCGAATCAATACCAAACCTTTA ATGAAGCCTTGATTGATTTGAAAAACGATCGAATTGATGGTCTATTGATTGACCG TGTCTATGCAAACTATTATTTAGAAGCAGAAGGTGTTTTAAACGATTATAATGTC TTTACAGTTGGACTAGAAACAGAAGCTTTTGCGGTTGGAGCCCGTAAGGAAGAT ACAAACTTGGTTAAGAAGATAAATGAAGCTTTTTCTAGTCTTTACAAGGACGGCA AGTTCCAAGAAATCAGCCAAAAATGGTTTGGAGAAGATGTAGCAACCAAAGAAG TAAAAGAAGGACAG

[0513] SEQ ID NO: 20, Rhavi-linker-PdT(G294P)-linker-SP0435 [aa 62-185] fusion protein, denoted SPP2:

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLT

GRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAI EQGQDTFQYVPTTENKSLLKDGGGGSSSANKAVNDFILAMNYDKKKLLTHQGESIE

NRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPT LLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNV

PARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTV SVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEV

EAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPSSGARVVTGKVDMVEDLIQE GSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVA QYYITWDELSYNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREGT GLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDGGGGSSSEQAIIETV PAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYGTEVIGVTV PTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVINTAEGTYVS R

[0514] SEQ ID NO : 21 , Rhavi-linker-PdT(G294P)-Iinker-SP0435 [aa 62- 185 ] fusion protein, denoted SPP2, includes leading methionine:

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYP LTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGP AIEQGQDTFQYVPTTENKSLLKDGGGGSSSANKAVNDFILAMNYDKKKLLTHQGES IENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENN PTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVN NVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIY YTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKS DEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPSSGARVVTGKVDMVED LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSG AYVAQYYITWDELSYNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKI REGTGLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDGGGGSSSEQA IIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYGTEVI GVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVINTAE GTYVSR

[0515] SEQ ID NO: 22, Rhavi-linker-PdT(G294P)-linker-SP0435 [aa 62-185]-Hisx6 fusion protein, denoted SPP2-H:

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLT GRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAI EQGQDTFQYVPTTENKSLLKDGGGGSSSANKAVNDFILAMNYDKKKLLTHQGESIE NRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPT LLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNV PARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTV SVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEV EAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPSSGARVVTGKVDMVEDLIQE GSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVA QYYITWDELSYNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREGT GLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDGGGGSSSEQAIIETV PAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYGTEVIGVTV PTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVINTAEGTYVS RHHHHHH

[0516] SEQ ID NO: 23, Rhavi-linker-PdT(G294P)-Iinker-SP0435 [aa 62-185]-Hisx6 fusion protein, denoted SPP2-H, includes leading methionine:

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYP LTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGP AIEQGQDTFQYVPTTENKSLLKDGGGGSSSANKAVNDFILAMNYDKKKLLTHQGES IENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENN PTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVN NVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIY YTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKS DEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPSSGARVVTGKVDMVED LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSG AYVAQYYITWDELSYNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKI REGTGLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDGGGGSSSEQA IIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYGTEVI GVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFTEAGQKLVINTAE GTYVSRHHHHHH

[0517] SEQ ID NO: 24, Rhavi-linker-SP0435 [aa 62-185]-linker-PdT(G294P) fusion protein:

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLT GRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAI EQGQDTFQYVPTTENKSLLKDGGGGSSSEQAIIETVPAQYLYKMDDTAYFMNTETY DQYEIPVVNVENELLYILENSDVKIQFYGTEVIGVTVPTTVELTVAETQPSIKGATVTG SGKPATMETGLVVNVPDFIEAGQKLVINTAEGTYVSRGGGGSSSANKAVNDFILAM NYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYP GALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVND LLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVH SGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAY GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPSSG ARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVT AYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAWDRNGQDLTAHFTTS IPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKV END

[0518] SEQ ID NO: 25, Rhavi-linker-SP0435 [aa 62-185]-linker-PdT(G294P)fusion protein, includes leading methionine:

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYP LTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGP AIEQGQDTFQYVPTTENKSLLKDGGGGSSSEQAIIETVPAQYLYKMDDTAYFMNTET YDQYEIPVVNVENELLYILENSDVKIQFYGTEVIGVTVPTTVELTVAETQPSIKGATVT GSGKPATMETGLVVNVPDFIEAGQKLVINTAEGTYVSRGGGGSSSANKAVNDFILA MNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRL YPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAV NDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNS VHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSV AYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPS SGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETK VTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPK WDRNGQDLTAHFT TSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVED KVEND

[0519] SEQ ID NO: 26, PdT(G294P)-linker-SP0435 [aa 62-185]-linker-Rhavi fusion protein:

ANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSD ISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVED PSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKT GNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGIS AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTE VKAVILGPDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATF QNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAWDR NGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIW GTTLYPQVEDKVENDGGGGSSSEQAIIETVPAQYLYKMDDTAYFMNTETYDQYEIP VVNVENELLYILENSDVKIQFYGTEVIGVTVPTTVELTVAETQPSIKGATVTGSGKPA TMETGLVVNVPDFIEAGQKLVINTAEGTYVSRGGGGSSSFDASNFKDFSSIASASSS WQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNN STENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENK SLLKD

[0520] SEQ ID NO: 27, SP0435 [aa 62-185]-linker-PdT(G294P)-linker-Rhavi fusion protein:

EQAIIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYG TEVIGVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVIN TAEGTYVSRGGGGSSSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPD EFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTY SIDLPGLAS SDSFLQVEDPSNS S VRG AVNDLLAKWHQD YGQVNNVP ARMQYEKITA HSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGD VFQDTVT VEDLKQRGIS AERPLVYIS S VA YGRQ VYLKLETTS KSDEVEAAFEALIKGV KVAPQTEWKQILDNTEVKAVILGPDPSSGARVVTGKVDMVEDLIQEGSRFTADHPG LPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELS YNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTV YEKTDLPLVRKRTISIWGTTLYPQVEDKVENDGGGGSSSFDASNFKDFSSIASASSSW QNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNST ENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSL LKD

[0521] SEQ ID NO: 28, PdT(G294P)-linker-Rhavi-linker-SP0435 [aa 62-185] fusion protein:

ANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSD ISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVED PSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKT GNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGIS AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTE VKAVILGPDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATF QNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPKAWDR NGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIW GTTLYPQVEDKVENDGGGGSSSFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFG NVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQ

VNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSEQAII

ETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYGTEVIG

VTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVINTAEGT

YVSR

[0522] SEQ ID NO : 29, SP0435 [aa 62- 185 ] -linker-Rhavi-linker-PdT(G294P) fusion protein:

EQAIIETVPAQYLYKMDDTAYFMNTETYDQYEIPVVNVENELLYILENSDVKIQFYG

TEVIGVTVPTTVELTVAETQPSIKGATVTGSGKPATMETGLVVNVPDFIEAGQKLVIN

TAEGTYVSRGGGGSSSFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQY

VNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNT

EIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSANKAVNDFILA

MNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRL

YPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAV

NDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNS

VHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSV

AYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGPDPS

SGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETK

VTAYRNGDLLLDHSGAYVAQYYITWDELSYNHQGKEVLTPK WDRNGQDLTAHFT

TSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVED

KVEND

[0523] SEQ ID NO: 30, Rhavi-linker-SP1500 [aa 27-278]-linker-SP0785 [aa 33-

399] fusion protein, denoted CPI:

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLT

GRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAI

EQGQDTFQYVPTTENKSLLKDGGGGSSSTSGDNWSKYQSNKSITIGFDSTFVPMGFA

QKDGSYAGFDIDLATAVFEKYGITVNWQPIDWDLKEAELTKGTIDLIWNGYSATDER

REKVAFSNSYMKNEQVLVTKKSSGITTAKDMTGKTLGAQAGSSGYADFEANPEILK

NIVANKEANQYQTFNEALIDLKNDRIDGLLIDRVYANYYLEAEGVLNDYNVFTVGLE

TEAFAVGARKEDTNLVKKINEAFSSLYKDGKFQEISQKWFGEDVATKEVKEGQAAA

FRQPSQTALKDEPTHLVVAKEGSVASSVLLSGTVTAKNEQYVYFDASKGDLDEILVS

VGDKVSEGQALVKYSSSEAQAAYDSASRAVARADRHINELNQARNEAASAPAPQLP APVGGEDATVQSPTPVAGNSVASIDAQLGDARDARADAAAQLSKAQSQLDATTVLS

TLEGTVVEVNSNVSKSPTGASQVMVHIVSNENLQVKGELSEYNLANLSVGQEVSFTS

KVYPDKKWTGKLSYISDYPKNNGEAASPAAGNNTGSKYPYTIDVTGEVGDLKQGFS

VNIEVKSKTKAILVPVSSLVMDDSKNYVWIVDEQQKAKKVEVSLGNADAENQEITS

GLTNGAKVISNPTS SLEEGKEVKADEATN

[0524] SEQ ID NO: 31, Rhavi-linker-SP1500 [aa 27-278]-linker-SP0785 [aa 33-

399] fusion protein, denoted CPI, includes leading methionine:

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYP

LTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGP

AIEQGQDTFQYVPTTENKSLLKDGGGGSSSTSGDNWSKYQSNKSITIGFDSTFVPMG

FAQKDGSYAGFDIDLATAVFEKYGITVNWQPIDWDLKEAELTKGTIDLIWNGYSATD

ERREKVAFSNSYMKNEQVLVTKKSSGITTAKDMTGKTLGAQAGSSGYADFEANPEI

LKNIVANKEANQYQTFNEALIDLKNDRIDGLLIDRVYANYYLEAEGVLNDYNVFTV

GLETEAFAVGARKEDTNLVKKINEAFSSLYKDGKFQEISQKWFGEDVATKEVKEGQ

AAAFRQPSQTALKDEPTHLVVAKEGSVASSVLLSGTVTAKNEQYVYFDASKGDLDE

ILVSVGDKVSEGQALVKYSSSEAQAAYDSASRAVARADRHINELNQARNEAASAPA

PQLPAPVGGEDATVQSPTPVAGNSVASIDAQLGDARDARADAAAQLSKAQSQLDAT

TVLSTLEGTVVEVNSNVSKSPTGASQVMVHIVSNENLQVKGELSEYNLANLSVGQE

VSFTSKVYPDKKWTGKLSYISDYPKNNGEAASPAAGNNTGSKYPYTIDVTGEVGDL

KQGFSVNIEVKSKTKAILVPVSSLVMDDSKNYVWIVDEQQKAKKVEVSLGNADAEN

QEITSGLTNGAKVISNPTSSLEEGKEVKADEATN

[0525] SEQ ID NO: 32, codon-optimized nucleotide sequence encoding Rhavi- linker-PdT(G294P)-linker-SP0435 [aa 62-185], denoted SPP2:

TTCGACGCATCCAACTTTAAAGACTTTAGCAGCATCGCGTCCGCAAGCTCTAGCT

GGCAGAATCAATCTGGTAGCACCATGATTATCCAAGTGGACAGCTTTGGTAACGT

CAGCGGTCAATATGTTAATCGTGCACAGGGTACGGGTTGTCAGAATTCTCCGTAC

CCGCTGACCGGTCGTGTTAACGGCACGTTCATCGCTTTCAGCGTCGGTTGGAACA

ATTCTACTGAAAATTGCAACAGCGCGACCGGTTGGACGGGCTATGCACAAGTGA

ATGGCAATAACACCGAAATCGTCACGTCCTGGAATCTGGCGTATGAGGGTGGCA

GCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGACCG

AGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGGCGAACAAGG

CGGTGAACGATTTTATCCTGGCGATGAACTATGACAAGAAGAAACTGCTGACCC ACCAAGGCGAGAGCATTGAGAACCGTTTCATTAAAGAAGGCAACCAGCTGCCGG

ACGAGTTTGTGGTTATCGAGCGTAAGAAACGTAGCCTGAGCACCAACACCAGCG

ACATTAGCGTGACCGCGACCAACGATAGCCGTCTGTACCCGGGTGCGCTGCTGGT

TGTGGATGAAACCCTGCTGGAAAACAACCCGACCCTGCTGGCGGTGGACCGTGC

GCCGATGACCTATAGCATCGATCTGCCGGGTCTGGCGAGCAGCGACAGCTTCCTG

CAAGTTGAGGATCCGAGCAACAGCAGCGTGCGTGGTGCGGTTAACGACCTGCTG

GCGAAGTGGCACCAGGATTACGGCCAAGTGAACAACGTTCCGGCGCGTATGCAG

TATGAAAAAATCACCGCGCACAGCATGGAGCAACTGAAGGTTAAATTCGGTAGC

GACTTTGAAAAGACCGGCAACAGCCTGGACATTGATTTCAACAGCGTGCACAGC

GGCGAGAAGCAGATCCAAATCGTTAACTTCAAGCAGATCTACTACACCGTGAGC

GTTGACGCGGTGAAGAACCCGGGTGACGTTTTCCAGGATACCGTGACCGTTGAA

GATCTGAAACAACGTGGCATTAGCGCGGAGCGTCCGCTGGTGTACATCAGCAGC

GTTGCGTACGGTCGTCAAGTGTATCTGAAGCTGGAAACCACCAGCAAAAGCGAT

GAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAGGGCGTGAAAGTTGCGCCGCAG

ACCGAATGGAAGCAAATTCTGGACAACACCGAGGTGAAAGCGGTTATTCTGGGC

CCGGATCCGAGCAGCGGCGCGCGTGTGGTTACCGGTAAAGTGGACATGGTTGAG

GATCTGATTCAGGAAGGTAGCCGTTTTACCGCGGACCACCCGGGCCTGCCGATCA

GCTACACCACCAGCTTCCTGCGTGACAACGTGGTTGCGACCTTTCAAAACAGCAC

CGATTACGTGGAAACCAAGGTTACCGCGTATCGTAACGGTGACCTGCTGCTGGA

CCACAGCGGTGCGTACGTGGCGCAGTACTATATCACCTGGGATGAACTGAGCTA

TAACCACCAGGGTAAAGAGGTGCTGACCCCGAAAGCGTGGGACCGTAACGGCCA

GGATCTGACCGCGCACTTCACCACCAGCATTCCGCTGAAGGGCAACGTGCGTAA

CCTGAGCGTTAAAATCCGTGAGGGTACCGGCCTGGCGTTTGAATGGTGGCGTACC

GTGTACGAGAAGACCGACCTGCCGCTGGTTCGTAAACGTACCATCAGCATTTGG

GGTACCACCCTGTATCCGCAGGTGGAGGACAAAGTTGAAAATGATGGTGGTGGT

GGTAGCAGCAGCGAGCAGGCGATCATTGAAACCGTGCCGGCGCAATACCTGTAT

AAGATGGACGATACCGCGTACTTCATGAACACCGAAACCTACGACCAATATGAA

ATTCCGGTGGTTAACGTTGAGAACGAACTGCTGTACATCCTGGAAAACAGCGAT

GTGAAAATTCAGTTTTATGGTACCGAGGTTATCGGCGTGACCGTTCCGACCACCG

TGGAGCTGACCGTTGCGGAAACCCAACCGAGCATCAAGGGTGCGACCGTGACCG

GTAGCGGTAAACCGGCGACCATGGAAACCGGTCTGGTGGTTAACGTGCCGGACT

TCATTGAGGCGGGCCAGAAGCTGGTTATCAATACCGCGGAGGGTACCTATGTTA

GCCGTTAATGA [0526] SEQ ID NO: 33, codon-optimized nucleotide sequence encoding Rhavi- linker-PdT(G294P)-linker-SP0435 [aa 62-185], denoted SPP2, includes leading ATG sequence:

ATGTTCGACGCATCCAACTTTAAAGACTTTAGCAGCATCGCGTCCGCAAGCTCTA GCTGGCAGAATCAATCTGGTAGCACCATGATTATCCAAGTGGACAGCTTTGGTAA CGTCAGCGGTCAATATGTTAATCGTGCACAGGGTACGGGTTGTCAGAATTCTCCG

TACCCGCTGACCGGTCGTGTTAACGGCACGTTCATCGCTTTCAGCGTCGGTTGGA ACAATTCTACTGAAAATTGCAACAGCGCGACCGGTTGGACGGGCTATGCACAAG TGAATGGCAATAACACCGAAATCGTCACGTCCTGGAATCTGGCGTATGAGGGTG

GCAGCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGA CCGAGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGGCGAACA AGGCGGTGAACGATTTTATCCTGGCGATGAACTATGACAAGAAGAAACTGCTGA

CCCACCAAGGCGAGAGCATTGAGAACCGTTTCATTAAAGAAGGCAACCAGCTGC CGGACGAGTTTGTGGTTATCGAGCGTAAGAAACGTAGCCTGAGCACCAACACCA GCGACATTAGCGTGACCGCGACCAACGATAGCCGTCTGTACCCGGGTGCGCTGC

TGGTTGTGGATGAAACCCTGCTGGAAAACAACCCGACCCTGCTGGCGGTGGACC GTGCGCCGATGACCTATAGCATCGATCTGCCGGGTCTGGCGAGCAGCGACAGCT TCCTGCAAGTTGAGGATCCGAGCAACAGCAGCGTGCGTGGTGCGGTTAACGACC

TGCTGGCGAAGTGGCACCAGGATTACGGCCAAGTGAACAACGTTCCGGCGCGTA TGCAGTATGAAAAAATCACCGCGCACAGCATGGAGCAACTGAAGGTTAAATTCG GTAGCGACTTTGAAAAGACCGGCAACAGCCTGGACATTGATTTCAACAGCGTGC

ACAGCGGCGAGAAGCAGATCCAAATCGTTAACTTCAAGCAGATCTACTACACCG TGAGCGTTGACGCGGTGAAGAACCCGGGTGACGTTTTCCAGGATACCGTGACCG TTGAAGATCTGAAACAACGTGGCATTAGCGCGGAGCGTCCGCTGGTGTACATCA

GCAGCGTTGCGTACGGTCGTCAAGTGTATCTGAAGCTGGAAACCACCAGCAAAA GCGATGAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAGGGCGTGAAAGTTGCGC CGCAGACCGAATGGAAGCAAATTCTGGACAACACCGAGGTGAAAGCGGTTATTC

TGGGCCCGGATCCGAGCAGCGGCGCGCGTGTGGTTACCGGTAAAGTGGACATGG TTGAGGATCTGATTCAGGAAGGTAGCCGTTTTACCGCGGACCACCCGGGCCTGCC GATCAGCTACACCACCAGCTTCCTGCGTGACAACGTGGTTGCGACCTTTCAAAAC

AGCACCGATTACGTGGAAACCAAGGTTACCGCGTATCGTAACGGTGACCTGCTG CTGGACCACAGCGGTGCGTACGTGGCGCAGTACTATATCACCTGGGATGAACTG AGCTATAACCACCAGGGTAAAGAGGTGCTGACCCCGAAAGCGTGGGACCGTAAC GGCCAGGATCTGACCGCGCACTTCACCACCAGCATTCCGCTGAAGGGCAACGTG

CGTAACCTGAGCGTTAAAATCCGTGAGGGTACCGGCCTGGCGTTTGAATGGTGG

CGTACCGTGTACGAGAAGACCGACCTGCCGCTGGTTCGTAAACGTACCATCAGC

ATTTGGGGTACCACCCTGTATCCGCAGGTGGAGGACAAAGTTGAAAATGATGGT

GGTGGTGGTAGCAGCAGCGAGCAGGCGATCATTGAAACCGTGCCGGCGCAATAC

CTGTATAAGATGGACGATACCGCGTACTTCATGAACACCGAAACCTACGACCAA

TATGAAATTCCGGTGGTTAACGTTGAGAACGAACTGCTGTACATCCTGGAAAAC

AGCGATGTGAAAATTCAGTTTTATGGTACCGAGGTTATCGGCGTGACCGTTCCGA

CCACCGTGGAGCTGACCGTTGCGGAAACCCAACCGAGCATCAAGGGTGCGACCG

TGACCGGTAGCGGTAAACCGGCGACCATGGAAACCGGTCTGGTGGTTAACGTGC

CGGACTTCATTGAGGCGGGCCAGAAGCTGGTTATCAATACCGCGGAGGGTACCT

ATGTTAGCCGTTAATGA

[0527] SEQ ID NO: 34, codon-optimized nucleotide sequence encoding Rhavi- linker-SP0435 [aa 62-185]-linker-PdT(G294P):

TTCGACGCATCCAACTTTAAAGACTTTAGCAGCATCGCGTCCGCAAGCTCTAGCT

GGCAGAATCAATCTGGTAGCACCATGATTATCCAAGTGGACAGCTTTGGTAACGT

CAGCGGTCAATATGTTAATCGTGCACAGGGTACGGGTTGTCAGAATTCTCCGTAC

CCGCTGACCGGTCGTGTTAACGGCACGTTCATCGCTTTCAGCGTCGGTTGGAACA

ATTCTACTGAAAATTGCAACAGCGCGACCGGTTGGACGGGCTATGCACAAGTGA

ATGGCAATAACACCGAAATCGTCACGTCCTGGAATCTGGCGTATGAGGGTGGCA

GCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGACCG

AGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGGAGCAGGCGA

TCATTGAAACCGTGCCGGCGCAATACCTGTATAAGATGGACGATACCGCGTACTT

CATGAACACCGAAACCTACGACCAATATGAAATTCCGGTGGTTAACGTTGAGAA

CGAACTGCTGTACATCCTGGAAAACAGCGATGTGAAAATTCAGTTTTATGGTACC

GAGGTTATCGGCGTGACCGTTCCGACCACCGTGGAGCTGACCGTTGCGGAAACC

CAACCGAGCATCAAGGGTGCGACCGTGACCGGTAGCGGTAAACCGGCGACCATG

GAAACCGGTCTGGTGGTTAACGTGCCGGACTTCATTGAGGCGGGCCAGAAGCTG

GTTATCAATACCGCGGAGGGTACCTATGTTAGCCGTTAATGAGGTGGTGGTGGTA

GCAGCAGCGCGAACAAGGCGGTGAACGATTTTATCCTGGCGATGAACTATGACA

AGAAGAAACTGCTGACCCACCAAGGCGAGAGCATTGAGAACCGTTTCATTAAAG

AAGGCAACCAGCTGCCGGACGAGTTTGTGGTTATCGAGCGTAAGAAACGTAGCC TGAGCACCAACACCAGCGACATTAGCGTGACCGCGACCAACGATAGCCGTCTGT

ACCCGGGTGCGCTGCTGGTTGTGGATGAAACCCTGCTGGAAAACAACCCGACCC

TGCTGGCGGTGGACCGTGCGCCGATGACCTATAGCATCGATCTGCCGGGTCTGGC

GAGCAGCGACAGCTTCCTGCAAGTTGAGGATCCGAGCAACAGCAGCGTGCGTGG

TGCGGTTAACGACCTGCTGGCGAAGTGGCACCAGGATTACGGCCAAGTGAACAA

CGTTCCGGCGCGTATGCAGTATGAAAAAATCACCGCGCACAGCATGGAGCAACT

GAAGGTTAAATTCGGTAGCGACTTTGAAAAGACCGGCAACAGCCTGGACATTGA

TTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAAATCGTTAACTTCAAGCA

GATCTACTACACCGTGAGCGTTGACGCGGTGAAGAACCCGGGTGACGTTTTCCA

GGATACCGTGACCGTTGAAGATCTGAAACAACGTGGCATTAGCGCGGAGCGTCC

GCTGGTGTACATCAGCAGCGTTGCGTACGGTCGTCAAGTGTATCTGAAGCTGGAA

ACCACCAGCAAAAGCGATGAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAGGGC

GTGAAAGTTGCGCCGCAGACCGAATGGAAGCAAATTCTGGACAACACCGAGGTG

AAAGCGGTTATTCTGGGCCCGGATCCGAGCAGCGGCGCGCGTGTGGTTACCGGT

AAAGTGGACATGGTTGAGGATCTGATTCAGGAAGGTAGCCGTTTTACCGCGGAC

CACCCGGGCCTGCCGATCAGCTACACCACCAGCTTCCTGCGTGACAACGTGGTTG

CGACCTTTCAAAACAGCACCGATTACGTGGAAACCAAGGTTACCGCGTATCGTA

ACGGTGACCTGCTGCTGGACCACAGCGGTGCGTACGTGGCGCAGTACTATATCA

CCTGGGATGAACTGAGCTATAACCACCAGGGTAAAGAGGTGCTGACCCCGAAAG

CGTGGGACCGTAACGGCCAGGATCTGACCGCGCACTTCACCACCAGCATTCCGCT

GAAGGGCAACGTGCGTAACCTGAGCGTTAAAATCCGTGAGGGTACCGGCCTGGC

GTTTGAATGGTGGCGTACCGTGTACGAGAAGACCGACCTGCCGCTGGTTCGTAA

ACGTACCATCAGCATTTGGGGTACCACCCTGTATCCGCAGGTGGAGGACAAAGT TGAAAATGAT

[0528] SEQ ID NO: 35, codon-optimized nucleotide sequence encoding Rhavi- linker-SP0435 [aa 62-185]-linker-PdT(G294P), includes leading ATG sequence:

ATGTTCGACGCATCCAACTTTAAAGACTTTAGCAGCATCGCGTCCGCAAGCTCTA

GCTGGCAGAATCAATCTGGTAGCACCATGATTATCCAAGTGGACAGCTTTGGTAA

CGTCAGCGGTCAATATGTTAATCGTGCACAGGGTACGGGTTGTCAGAATTCTCCG

TACCCGCTGACCGGTCGTGTTAACGGCACGTTCATCGCTTTCAGCGTCGGTTGGA

ACAATTCTACTGAAAATTGCAACAGCGCGACCGGTTGGACGGGCTATGCACAAG

TGAATGGCAATAACACCGAAATCGTCACGTCCTGGAATCTGGCGTATGAGGGTG GCAGCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGA

CCGAGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGGAGCAGG

CGATCATTGAAACCGTGCCGGCGCAATACCTGTATAAGATGGACGATACCGCGT

ACTTCATGAACACCGAAACCTACGACCAATATGAAATTCCGGTGGTTAACGTTGA

GAACGAACTGCTGTACATCCTGGAAAACAGCGATGTGAAAATTCAGTTTTATGGT

ACCGAGGTTATCGGCGTGACCGTTCCGACCACCGTGGAGCTGACCGTTGCGGAA

ACCCAACCGAGCATCAAGGGTGCGACCGTGACCGGTAGCGGTAAACCGGCGACC

ATGGAAACCGGTCTGGTGGTTAACGTGCCGGACTTCATTGAGGCGGGCCAGAAG

CTGGTTATCAATACCGCGGAGGGTACCTATGTTAGCCGTTAATGAGGTGGTGGTG

GTAGCAGCAGCGCGAACAAGGCGGTGAACGATTTTATCCTGGCGATGAACTATG

ACAAGAAGAAACTGCTGACCCACCAAGGCGAGAGCATTGAGAACCGTTTCATTA

AAGAAGGCAACCAGCTGCCGGACGAGTTTGTGGTTATCGAGCGTAAGAAACGTA

GCCTGAGCACCAACACCAGCGACATTAGCGTGACCGCGACCAACGATAGCCGTC

TGTACCCGGGTGCGCTGCTGGTTGTGGATGAAACCCTGCTGGAAAACAACCCGA

CCCTGCTGGCGGTGGACCGTGCGCCGATGACCTATAGCATCGATCTGCCGGGTCT

GGCGAGCAGCGACAGCTTCCTGCAAGTTGAGGATCCGAGCAACAGCAGCGTGCG

TGGTGCGGTTAACGACCTGCTGGCGAAGTGGCACCAGGATTACGGCCAAGTGAA

CAACGTTCCGGCGCGTATGCAGTATGAAAAAATCACCGCGCACAGCATGGAGCA

ACTGAAGGTTAAATTCGGTAGCGACTTTGAAAAGACCGGCAACAGCCTGGACAT

TGATTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAAATCGTTAACTTCAA

GCAGATCTACTACACCGTGAGCGTTGACGCGGTGAAGAACCCGGGTGACGTTTT

CCAGGATACCGTGACCGTTGAAGATCTGAAACAACGTGGCATTAGCGCGGAGCG

TCCGCTGGTGTACATCAGCAGCGTTGCGTACGGTCGTCAAGTGTATCTGAAGCTG

GAAACCACCAGCAAAAGCGATGAGGTTGAAGCGGCGTTTGAGGCGCTGATTAAG

GGCGTGAAAGTTGCGCCGCAGACCGAATGGAAGCAAATTCTGGACAACACCGAG

GTGAAAGCGGTTATTCTGGGCCCGGATCCGAGCAGCGGCGCGCGTGTGGTTACC

GGTAAAGTGGACATGGTTGAGGATCTGATTCAGGAAGGTAGCCGTTTTACCGCG

GACCACCCGGGCCTGCCGATCAGCTACACCACCAGCTTCCTGCGTGACAACGTG

GTTGCGACCTTTCAAAACAGCACCGATTACGTGGAAACCAAGGTTACCGCGTATC

GTAACGGTGACCTGCTGCTGGACCACAGCGGTGCGTACGTGGCGCAGTACTATA

TCACCTGGGATGAACTGAGCTATAACCACCAGGGTAAAGAGGTGCTGACCCCGA

AAGCGTGGGACCGTAACGGCCAGGATCTGACCGCGCACTTCACCACCAGCATTC

CGCTGAAGGGCAACGTGCGTAACCTGAGCGTTAAAATCCGTGAGGGTACCGGCC TGGCGTTTGAATGGTGGCGTACCGTGTACGAGAAGACCGACCTGCCGCTGGTTCG

TAAACGTACCATCAGCATTTGGGGTACCACCCTGTATCCGCAGGTGGAGGACAA

AGTTGAAAATGAT

[0529] SEQ ID NO: 36, codon-optimized nucleic acid sequence encoding Rhavi- linker-SPl 500 [aa 27-278]-linker-SP0785 [aa 33-399] fusion protein, denoted CPI :

TTCGACGCATCCAACTTTAAAGACTTTAGCAGCATCGCGTCCGCAAGCTCTAGCT

GGCAGAATCAATCTGGTAGCACCATGATTATCCAAGTGGACAGCTTTGGTAACGT

CAGCGGTCAATATGTTAATCGTGCACAGGGTACGGGTTGTCAGAATTCTCCGTAC

CCGCTGACCGGTCGTGTTAACGGCACGTTCATCGCTTTCAGCGTCGGTTGGAACA

ATTCTACTGAAAATTGCAACAGCGCGACCGGTTGGACGGGCTATGCACAAGTGA

ATGGCAATAACACCGAAATCGTCACGTCCTGGAATCTGGCGTATGAGGGTGGCA

GCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGACCG

AGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGACCAGCGGCG

ACAATTGGTCCAAATACCAGAGCAACAAGAGCATCACGATCGGCTTCGACAGCA

CTTTTGTGCCGATGGGTTTCGCGCAAAAAGACGGTAGCTACGCGGGTTTCGATAT

TGACCTGGCGACCGCTGTCTTTGAGAAATACGGCATTACGGTTAATTGGCAGCCG

ATTGATTGGGACCTGAAAGAGGCCGAACTCACCAAAGGCACCATCGACCTGATC

TGGAATGGTTACTCCGCAACCGATGAGCGTCGCGAAAAAGTTGCCTTCAGCAAC

AGCTATATGAAGAATGAACAAGTGTTGGTAACCAAGAAATCTAGCGGCATTACG

ACCGCGAAAGACATGACCGGTAAGACGCTGGGTGCGCAGGCCGGTAGCTCTGGC

TATGCGGATTTCGAGGCGAATCCTGAGATTCTGAAAAACATCGTTGCGAATAAA

GAGGCGAACCAGTACCAGACCTTTAACGAAGCACTGATCGACCTGAAAAACGAT

CGCATTGACGGTCTGCTGATCGATCGTGTGTACGCGAACTATTATCTGGAAGCCG

AGGGCGTTCTGAACGATTATAATGTTTTTACCGTGGGTCTGGAGACTGAGGCATT

CGCGGTTGGTGCGCGCAAGGAAGATACCAACCTGGTTAAAAAGATTAATGAGGC

ATTTAGCTCACTGTACAAGGACGGCAAGTTCCAAGAAATTAGCCAGAAGTGGTT

CGGTGAAGATGTTGCGACGAAAGAGGTTAAAGAGGGCCAAGCGGCCGCATTTCG

CCAACCGAGCCAGACTGCGTTGAAAGATGAGCCGACCCATCTGGTTGTTGCGAA

AGAGGGCAGCGTGGCATCGAGCGTGCTGCTGAGCGGTACGGTTACTGCCAAAAA

CGAACAATACGTGTACTTCGATGCTAGCAAGGGTGATCTGGATGAAATTCTGGTG

AGCGTGGGTGACAAAGTTAGCGAAGGCCAGGCACTGGTGAAGTATTCATCCTCC GAGGCACAGGCAGCGTACGACAGCGCAAGCCGCGCAGTGGCGCGTGCCGACCGT CACATTAACGAATTGAACCAAGCGCGTAACGAGGCCGCAAGCGCGCCAGCACCG CAGCTGCCGGCTCCGGTGGGTGGCGAAGATGCGACGGTGCAGAGCCCGACCCCG GTTGCGGGTAATTCGGTCGCCAGCATCGATGCGCAGCTGGGTGACGCGCGTGAT

GCCCGTGCGGATGCGGCTGCTCAACTGAGCAAGGCTCAGAGCCAACTGGACGCG ACGACGGTGCTGAGCACCTTGGAGGGTACCGTTGTCGAAGTCAACAGCAATGTG AGCAAGAGCCCAACGGGTGCGAGCCAGGTTATGGTCCACATTGTGAGCAATGAA

AACTTACAGGTCAAGGGTGAGCTGAGCGAGTATAACCTGGCGAATCTGAGCGTT GGTCAAGAGGTCAGCTTTACCAGCAAGGTCTACCCGGATAAGAAATGGACCGGC AAGTTGAGCTACATCAGCGACTACCCGAAGAACAATGGCGAGGCAGCCTCCCCG

GCAGCCGGCAACAATACCGGCTCTAAGTATCCGTACACCATCGACGTAACCGGT

GAGGTCGGCGACCTGAAACAGGGTTTTAGCGTGAATATCGAAGTGAAGTCCAAG ACCAAGGCAATTTTGGTTCCGGTTAGCTCCCTGGTGATGGACGATAGCAAGAATT ATGTGTGGATTGTCGACGAGCAACAGAAAGCGAAAAAAGTTGAAGTGAGCCTGG GCAATGCTGATGCCGAGAACCAAGAAATCACGTCTGGTCTGACCAACGGTGCGA AAGTTATTAGCAACCCGACCAGCAGCCTGGAAGAGGGTAAAGAGGTCAAAGCCG

ACGAAGCTACGAAC

[0530] SEQ ID NO: 37, codon-optimized nucleic acid sequence encoding Rhavi- linker-SP1500 [aa 27-278]-linker-SP0785 [aa 33-399] fusion protein, denoted CPI, includes leading ATG sequence:

GCAGCGGTCCGGCTATTGAACAGGGCCAGGATACCTTCCAATACGTCCCTACGA CCGAGAATAAGTCCCTTCTGAAAGACGGCGGTGGCGGTTCGAGCTCGACCAGCG GCGACAATTGGTCCAAATACCAGAGCAACAAGAGCATCACGATCGGCTTCGACA

GCACTTTTGTGCCGATGGGTTTCGCGCAAAAAGACGGTAGCTACGCGGGTTTCGA TATTGACCTGGCGACCGCTGTCTTTGAGAAATACGGCATTACGGTTAATTGGCAG CCGATTGATTGGGACCTGAAAGAGGCCGAACTCACCAAAGGCACCATCGACCTG

ATCTGGAATGGTTACTCCGCAACCGATGAGCGTCGCGAAAAAGTTGCCTTCAGC AACAGCTATATGAAGAATGAACAAGTGTTGGTAACCAAGAAATCTAGCGGCATT

ACGACCGCGAAAGACATGACCGGTAAGACGCTGGGTGCGCAGGCCGGTAGCTCT

GGCTATGCGGATTTCGAGGCGAATCCTGAGATTCTGAAAAACATCGTTGCGAAT

AAAGAGGCGAACCAGTACCAGACCTTTAACGAAGCACTGATCGACCTGAAAAAC

GATCGCATTGACGGTCTGCTGATCGATCGTGTGTACGCGAACTATTATCTGGAAG

CCGAGGGCGTTCTGAACGATTATAATGTTTTTACCGTGGGTCTGGAGACTGAGGC

ATTCGCGGTTGGTGCGCGCAAGGAAGATACCAACCTGGTTAAAAAGATTAATGA

GGCATTTAGCTCACTGTACAAGGACGGCAAGTTCCAAGAAATTAGCCAGAAGTG

GTTCGGTGAAGATGTTGCGACGAAAGAGGTTAAAGAGGGCCAAGCGGCCGCATT

TCGCCAACCGAGCCAGACTGCGTTGAAAGATGAGCCGACCCATCTGGTTGTTGC

GAAAGAGGGCAGCGTGGCATCGAGCGTGCTGCTGAGCGGTACGGTTACTGCCAA

AAACGAACAATACGTGTACTTCGATGCTAGCAAGGGTGATCTGGATGAAATTCT

GGTGAGCGTGGGTGACAAAGTTAGCGAAGGCCAGGCACTGGTGAAGTATTCATC

CTCCGAGGCACAGGCAGCGTACGACAGCGCAAGCCGCGCAGTGGCGCGTGCCGA

CCGTCACATTAACGAATTGAACCAAGCGCGTAACGAGGCCGCAAGCGCGCCAGC

ACCGCAGCTGCCGGCTCCGGTGGGTGGCGAAGATGCGACGGTGCAGAGCCCGAC

CCCGGTTGCGGGTAATTCGGTCGCCAGCATCGATGCGCAGCTGGGTGACGCGCG

TGATGCCCGTGCGGATGCGGCTGCTCAACTGAGCAAGGCTCAGAGCCAACTGGA

CGCGACGACGGTGCTGAGCACCTTGGAGGGTACCGTTGTCGAAGTCAACAGCAA

TGTGAGCAAGAGCCCAACGGGTGCGAGCCAGGTTATGGTCCACATTGTGAGCAA

TGAAAACTTACAGGTCAAGGGTGAGCTGAGCGAGTATAACCTGGCGAATCTGAG

CGTTGGTCAAGAGGTCAGCTTTACCAGCAAGGTCTACCCGGATAAGAAATGGAC

CGGCAAGTTGAGCTACATCAGCGACTACCCGAAGAACAATGGCGAGGCAGCCTC

CCCGGCAGCCGGCAACAATACCGGCTCTAAGTATCCGTACACCATCGACGTAAC

CGGTGAGGTCGGCGACCTGAAACAGGGTTTTAGCGTGAATATCGAAGTGAAGTC

CAAGACCAAGGCAATTTTGGTTCCGGTTAGCTCCCTGGTGATGGACGATAGCAA

GAATTATGTGTGGATTGTCGACGAGCAACAGAAAGCGAAAAAAGTTGAAGTGAG

CCTGGGCAATGCTGATGCCGAGAACCAAGAAATCACGTCTGGTCTGACCAACGG

TGCGAAAGTTATTAGCAACCCGACCAGCAGCCTGGAAGAGGGTAAAGAGGTCAA

AGCCGACGAAGCTACGAAC

[0531] SEQ ID NO: 38, linker sequence [7 amino acids]:

GGGGSSS [0532] SEQ ID NO: 39, linker sequence [5 amino acid repeats]:

(GGGGS)_n

[0533] SEQ ID NO: 40, linker sequence [5 amino acids]:

GGGGS

[0534] SEQ ID NO: 41, linker sequence [15 amino acids]:

GGGGSGGGGSGGGGS

[0535] SEQ ID NO: 42, linker sequence [30 amino acids]:

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

[0536] SEQ ID NO: 43, linker sequence [18 amino acids]:

KESGSVSSEQLAQFRSLD

[0537] SEQ ID NO: 44, linker sequence [14 amino acids]:

EGKSSGSGSESKST

[0538] SEQ ID NO: 45, linker sequence:

(Gly)n

[0539] SEQ ID NO: 46, linker sequence [6 amino acids]:

GGGGGG

[0540] SEQ ID NO: 47, linker sequence [8 amino acids]:

GGGGGGGG

[0541] SEQ ID NO: 48, linker sequence [12 amino acids]:

GSAGSAAGSGEF

[0542] SEQ ID NO: 49, linker sequence [5 amino acid repeats]:

(EAAAK)n

[0543] SEQ ID NO: 50, linker sequence:

EAAAK

[0544] SEQ ID NO: 51, linker sequence:

A(EAAAK)„A [0545] SEQ ID NO: 52, linker sequence:

A(EAAAK)_nA , n=l-5

[0546] SEQ ID NO: 53, linker sequence:

A(EAAAK)₄ALEA(EAAAK)₄A

[0547] SEQ ID NO: 54, linker sequence:

[A(EAAAK)_nA]m , n=l-4, m=l-2

[0548] SEQ ID NO: 55, linker sequence [12 amino acids]:

AEAAAKEAAAKA

[0549] SEQ ID NO: 56, linker sequence [2 amino acid repeats]:

(XP)_n

[0550] SEQ ID NO: 57, linker sequence [2 amino acid repeats]:

(AP)„

[0551] SEQ ID NO: 58, linker sequence [2 amino acid repeats]:

(AP)_n , n=l-17

[0552] SEQ ID NO: 59, linker sequence [2 amino acid repeats]:

(KP)_n

[0553] SEQ ID NO: 60, linker sequence [2 amino acid repeats]:

(QP)n

[0554] SEQ ID NO: 61, linker sequence [14 amino acids]:

APAPAPAPAPAPAP

[0555] SEQ ID NO: 62, GAG linker sequence [21 amino acids]:

GAPGGGGGAAAAAGGGGGGAP

[0556] SEQ ID NO: 63, GAG2 linker sequence [39 amino acids]:

GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP

[0557] SEQ ID NO: 64, GAG3 linker sequence [57 amino acids]: GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGG

GGGAP

[0558] SEQ ID NO: 65, linker sequence [4 amino acids]:

VSDP

[0559] SEQ ID NO: 66, linker sequence [4 amino acids]:

AAAA

[0560] SEQ ID NO: 67, linker sequence [4 amino acids]:

GGGG

[0561] SEQ ID NO: 68, His tag 1 :

(His)n , n=2-10

[0562] SEQ ID NO: 69, His tag 2:

HHHHHH

[0563] SEQ ID NO: 70, His tag 3:

MSYYHHHHHH

[0564] SEQ ID NO: 71 , signal peptide sequence:

MKKIWLALAGLVLAFSASA

[0565] SEQ ID NO: 72, signal peptide sequence:

MAPFEPLASGILLLLWLIAPSRA

[0566] SEQ ID NO: 73, signal peptide sequence:

MKKVAAFVALSLLMAGC

[0567] SEQ ID NO: 74, signal peptide sequence:

MKKIMLVITLILVSPIAQQTEAKD

[0568] SEQ ID NO: 75, signal peptide sequence:

MKKKIISAILMSTVILSAAAPLSGVYADT

[0569] SEQ ID NO: 76, signal peptide sequence:

MKKRKVLIPLMALSTIL VS STGNLEVIQ AEV [0570] SEQ ID NO: 77, signal peptide sequence:

MNMKKATIAATAGIAVTAFAAPTIASAST

[0571] SEQ ID NO: 78, signal peptide sequence:

MQKTRKERILEALQEEKKNKKSKKFKTGATIAGVTAIATSITVPGIEVIVSADE

[0572] SEQ ID NO: 79, signal peptide sequence:

MKKLKMASCALVAGLMFSGLTPNAFAED

[0573] SEQ ID NO: 80, signal peptide sequence:

MAKKFNYKLPSMVALTLVGSAVTAHQVQAAE

[0574] SEQ ID NO: 81, signal peptide sequence:

MTDKKSENQTEKTETKENKGMTRREMLKLSAVAGTGIAVGATGLGTILNVVDQVD

KALT

[0575] SEQ ID NO: 82, signal peptide sequence:

MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLGLGLTIAQSVGAFG

[0576] SEQ ID NO: 83, signal peptide sequence:

MEFGLSWLFLVAILKGVQC

[0577] SEQ ID NO: 84, signal peptide sequence:

MGWSCIILFLVATATGVHS

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Equivalents

[0578] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

Claims We claim:

(a) a biotinylated polysaccharide antigen; and

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

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.

(a) a biotinylated polysaccharide antigen; and

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

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 about

1.25 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, 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.

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: 1

20. 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;

(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 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

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:

(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:

(b) a salt, optionally wherein the salt is or comprises sodium chloride;

(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.