Immunogenic components

Abstract

A technique for providing an immunogenic composition (e.g., a vaccine) and a method for inducing an immune response in a subject requiring the same.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 452,681, filed on March 16, 2023, the entire content of which is incorporated herein by reference.

Background Art

[0002] Most of the vaccines manufactured worldwide each year are administered to infants, children, and adults. Typically, vaccines contain excipients, some of which may be added for specific purposes. Specific common excipients present in vaccines include preservatives, adjuvants, stabilizers, and the like.

Summary of the Invention

Problems to be Solved by the Invention

[0003] As an example, generally, adjuvants are added to help stimulate a stronger immune response. Adjuvants containing aluminum have been components of vaccines used since the 1930s. Surprisingly, despite their widespread use, the mechanism regarding the immunostimulatory properties of aluminum - based adjuvants remains unclear.

Means for Solving the Problems

[0004] In particular, the present disclosure provides insights and techniques useful for providing immunogenic compositions (e.g., vaccines). In some embodiments, the insights and techniques provided herein may be useful for providing immunogenic compositions (e.g., vaccines) with enhanced immunogenicity to prevent specific diseases or disorders. In certain embodiments, the insights and techniques described herein are useful for MAPS - based immunogenic compositions (e.g., vaccines).

[0005] The applicant has previously utilized MAPS technology to develop polyvalent vaccines in which at least one polysaccharide antigen is non-covalently bound to one or more polypeptide antigens, such as polyvalent pneumococcal vaccines as described in WO 2020 / 056202 and PCT / US2022 / 043156 (for the purposes described herein, the relevant content is incorporated herein by reference in its entirety).

[0006] In particular, this disclosure provides the insight that the immunogenicity of MAPS immunogenic compositions can be improved by changing the type and / or concentration of certain excipients (including, but not limited to, adjuvants) and / or formulation buffers used in the MAPS immunogenic compositions. For example, in one embodiment, aluminum-based adjuvants are widely used in vaccines to enhance the ability to elicit an immune response to the vaccine and to improve the overall efficacy of the vaccine, but this disclosure provides the insight that, in particular with respect to MAPS immunogenic compositions, it may be desirable to reduce the content of aluminum-based adjuvants. Without wishing to adhere to any particular theory, the reduction in the content of aluminum-based adjuvants is characterized in some embodiments by the amount of aluminum present in the MAPS immunogenic composition, while in other embodiments it is characterized by the ratio of the aluminum content to the polysaccharide antigen content and / or polypeptide antigen content. In certain embodiments, the Disclosure demonstrates that MAPS immunogenic compositions comprising an aluminum-based adjuvant at an aluminum concentration of less than 1.25 mg / mL in the composition may be particularly useful in increasing the immune response to polysaccharide antigens (multiple) and / or polypeptide antigens (multiple) (e.g., in some embodiments, B-cell immune responses and / or T-cell immune responses). In some embodiments, the Disclosure demonstrates that MAPS immunogenic compositions comprising an aluminum-based adjuvant, wherein the ratio of the mass of aluminum in the composition (derived from the aluminum-based adjuvant) to the total mass of MAPS-derived polysaccharide antigens (multiple) is 3.3:1, may be particularly useful in increasing the immune response to polysaccharide antigens (multiple) and / or polypeptide antigens (multiple) (e.g., in some embodiments, B-cell immune responses and / or T-cell immune responses).In some embodiments, the disclosure demonstrates that a MAPS immunogenic composition comprising an aluminum-based adjuvant, wherein the ratio of the mass of aluminum in the composition (derived from the aluminum-based adjuvant) to the total mass of MAPS-derived fusion proteins (multiple) is 1.1:1, may be particularly useful in increasing the immune response to polysaccharide antigens (multiple) and / or polypeptide antigens (multiple) (for example, in some embodiments, B-cell immune responses and / or T-cell immune responses). In some embodiments, the disclosure demonstrates that a MAPS immunogenic composition comprising an aluminum-based adjuvant, wherein the ratio of the mass of aluminum in the composition (derived from the aluminum-based adjuvant) to the total mass of MAPS immunogenic complexes is 0.8:1, may be particularly useful in increasing the immune response to polysaccharide antigens (multiple) and / or polypeptide antigens (multiple) (for example, in some embodiments, B-cell immune responses and / or T-cell immune responses).

[0007] In particular, and without intending to confine ourselves to any specific theory, this disclosure offers the insight that the presence of MAPS complexes in a free form (i.e., MAPS complexes not associated with aluminum-based adjuvants) may be desirable for inducing an immune response to an antigen compared to MAPS complexes associated with aluminum-based adjuvants.

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

[0009] In some embodiments, the disclosure suggests that certain formulation buffers may be more desirable for use with MAPS immunogenic compositions (e.g., vaccines). In some embodiments, succinate buffers may be desired for use with such MAPS immunogenic compositions.

[0010] Accordingly, this disclosure provides, in particular, technologies (e.g., compositions, methods, and / or kits) for inducing an immune response in subjects requiring them. In certain 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 immunogenic complexes, wherein at least one of the immunogenic complexes comprises (a) a biotinylated polysaccharide antigen and (b) a fusion protein comprising (i) a biotin-binding site and (ii) at least one polypeptide antigen, wherein the biotinylated polysaccharide antigen is non-covalently bound to the biotin-binding site of the fusion protein. In some embodiments, the pharmaceutical composition further comprises an aluminum-based adjuvant or an adjuvant comprising an aluminum-based adjuvant in small amounts of aluminum.

[0012] Other aspects of the present disclosure provide methods for producing the pharmaceutical compositions described herein. For example, in some embodiments, such methods include the step of combining an immunogenic composition with an aluminum-based adjuvant or an adjuvant containing an aluminum-based adjuvant to form a pharmaceutical composition having a small amount of aluminum, wherein the immunogenic composition comprises one or more immunogenic complexes, at least one of which comprises a fusion protein comprising (a) a biotinylated polysaccharide antigen and (b)(i) a biotin-binding site and (ii) at least one antigenic polypeptide, wherein the biotinylated polysaccharide antigen is non-covalently bound to the biotin-binding site of the fusion protein.

[0013] In some embodiments, the reduction in the amount of aluminum in the pharmaceutical compositions described herein may be expressed as the mass of aluminum element (derived from an aluminum-based adjuvant) relative to the solution volume of each pharmaceutical composition. In some embodiments, the reduction in the amount of aluminum in the pharmaceutical compositions described herein may be reflected by the ratio of the mass or molar amount of aluminum element present in the pharmaceutical composition to the total mass or total molar amount of biotinylated polysaccharide antigen(s). In some embodiments, the reduction in the amount of aluminum in the pharmaceutical compositions described herein may be reflected by the ratio of the mass or molar amount of aluminum element present in the pharmaceutical composition to the total mass or total molar amount of fusion protein(s). In some embodiments, the reduction in the amount of aluminum in the pharmaceutical compositions described herein may be reflected by the ratio of the mass or molar amount of aluminum element present in the pharmaceutical composition to the total mass or total molar amount of the immunogenic complex(s).

[0014] In some embodiments of the various aspects described herein, the aluminum concentration in the pharmaceutical composition is reduced relative to the reference composition. In some embodiments, the pharmaceutical composition, upon administration to a subject, is characterized by inducing an immune response to (i) at least one polysaccharide antigen and / or (ii) at least one polypeptide antigen at a level higher than the corresponding reference level, where the reference level is the level of immune response induced in the subject after administration of the reference composition. In some embodiments, the immune response is or includes an antibody response and / or a B-cell response.

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

[0016] In some embodiments of the various aspects described herein, the aluminum concentration in the pharmaceutical composition is less than 1.25 mg / mL, for example, about 0.125 mg / mL to about 1.25 mg / mL. In some embodiments, the aluminum concentration 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 aluminum concentration in the pharmaceutical composition is less than 0.25 mg / mL, for example, about 0.125 mg / mL to about 0.25 mg / mL. In some embodiments, the aluminum concentration in the pharmaceutical composition is about 0.35 mg / mL to about 0.65 mg / mL. In some embodiments, the aluminum concentration in the pharmaceutical composition is about 0.4 to about 0.6 mg / mL. In some embodiments, the aluminum concentration in the pharmaceutical composition is about 0.5 mg / mL. In some embodiments, the aluminum concentration in the pharmaceutical composition is at least 0.35 mg / mL. In some embodiments, the aluminum concentration in the pharmaceutical composition is at least 0.25 mg / mL. In some embodiments, the aluminum concentration 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 the various aspects described herein, the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is less than 3.68:1. In some embodiments, the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen 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 in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is about 1.91:1 to about 1.03:1. In some embodiments, the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen 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 in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is about 1.47:1. In some embodiments, the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is at least 1.03:1. In some embodiments, the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is at least 0.73:1.

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

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

[0020] In some embodiments of the various aspects described herein, the aluminum-based adjuvant contained in the pharmaceutical composition described herein is aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate (alum), or a combination thereof, or contains thereof. In some embodiments, the aluminum-based adjuvant is aluminum phosphate. In some embodiments, the concentration of aluminum phosphate in the pharmaceutical composition is less than 5.65 mg / mL. In some embodiments, the concentration of 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 aluminum phosphate in the pharmaceutical composition is at least 1.58 mg / mL. In some embodiments, the concentration of aluminum phosphate in the pharmaceutical composition is at least 1.13 mg / mL.

[0021] In some embodiments of the various aspects described herein, the biotinylated polysaccharide antigens contained in the immunogenic complex described herein include bacterial polysaccharide antigens, fungal polysaccharide antigens, parasitic polysaccharide antigens, viral polysaccharide antigens, and mammalian polysaccharide antigens (e.g., tumor polysaccharide antigens). In some embodiments, the biotinylated polysaccharide antigens include polysaccharide antigens associated with pathogens (e.g., bacteria, fungi, parasites, or viruses). In some embodiments, the biotinylated polysaccharide antigens include polysaccharide antigens associated with tumors. In some embodiments, the polysaccharide antigen is a polysaccharide selected from the group consisting of Salmonella polysaccharide, pneumococcal polysaccharide, Haemophili polysaccharide, meningococcal polysaccharide, staphylococcal polysaccharide, Bacillus anthracis polysaccharide, Streptococcus polysaccharide, Pseudomonas polysaccharide, Klebsiella polysaccharide, Cryptococcus polysaccharide, polysaccharides of the capsular or cell wall of other bacteria, viral polysaccharides (e.g., viral glycoproteins), or a combination thereof, or comprising such polysaccharides.

[0022] In some embodiments of the various aspects described herein, the biotinylated polysaccharide antigen contained in the immunogenic complex described herein contains one or more polysaccharides derived from Streptococcus pneumoniae. In some embodiments, the biotinylated polysaccharide antigens are 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20 Contains one or more polysaccharides of Streptococcus pneumoniae, each having a serotype independently selected from A, 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. In some embodiments, the biotinylated polysaccharide antigen contains one or more polysaccharides of Streptococcus pneumoniae having serotypes 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.

[0023] In some embodiments of the various aspects described herein, at least one polypeptide antigen(s) contained in the immunogenic complex described herein is or includes 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 polypeptide antigen(s) is or includes a polypeptide antigen associated with a pathogen (e.g., bacteria, fungi, parasites, or viruses). In some embodiments, at least one polypeptide antigen(s) is or includes a polypeptide antigen associated with a tumor.

[0024] In some embodiments of the various aspects described herein, at least one of the polypeptide antigens (may be multiple) contained in the immunogenic complex described herein is a streptococcal antigen (e.g., antigens of Streptococcus pneumoniae, group A, group B, and viridans), tuberculosis antigen, tetanus antigen, anthrax antigen, pertussis antigen, staphylococcal antigen (e.g., Staphylococcus aureus), Haemophilus antigen, Enterobacter antigen, Acinetobacter antigen, Citrobacter antigen, Serratia antigen, Clostridium antigen, Campylobacter antigen, Vibriocholera antigen, Pseudomonas antigen, Meningococcal antigen, Neisseria gonorrhoeae Antigens selected from the group consisting of gonorrhoeae antigen, Chlamydia trachomatis antigen, Klebsiella antigen, Shigella antigen, Salmonella antigen, Escherichia coli antigen, malaria antigen, HIV antigen, HPV antigen, influenza (e.g., seasonal or epidemic) antigen, coronavirus antigen (e.g., SARS-CoV-2 antigen), herpes (e.g., HSV) antigen, tumor antigen, and combinations thereof or comprising such antigens. In some embodiments, at least one polypeptide antigen is or comprises pneumococcal polypeptide antigen. In some embodiments, at least one polypeptide antigen is or comprises pneumococcal polypeptide antigen, SP1500 polypeptide antigen, SP0785 polypeptide antigen, SP0435 polypeptide antigen, or combinations thereof.

[0025] In some embodiments of the various aspects described herein, at least one immunogenic complex contained in the pharmaceutical composition described herein comprises a fusion protein comprising (i) a biotin-binding site, (ii) a pneumolysin polypeptide antigen or an antigenic fragment thereof, and (iii) an SP0435 polypeptide antigen or an antigenic fragment thereof. In some embodiments, the fusion protein has or comprises an amino acid sequence that is 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 the various aspects described herein, at least one immunogenic complex contained in the pharmaceutical composition described herein comprises a fusion protein comprising (i) a biotin-binding site, (ii) an SP1500 polypeptide antigen or its antigenic fragment, and (iii) an SP0785 polypeptide antigen or its antigenic fragment. In some embodiments, the fusion protein has or contains an amino acid sequence that is 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 the various aspects described herein, the immunogenic composition contained in the pharmaceutical composition 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 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 immunogenic complexes. In some embodiments, the immunogenic composition comprises 60 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, or 35 or fewer immunogenic complexes.

[0028] In some embodiments of the various aspects described herein, one or more immunogenic complexes contained in the pharmaceutical composition described herein are (i) a first plurality of immunogenic complexes comprising a biotinylated polysaccharide antigen selected from the serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F of Streptococcus pneumoniae, wherein each biotinylated polysaccharide antigen is non-covalently complexed with a biotin-binding site of a first fusion protein, where the first The fusion protein contains (a) a biotin binding site, (b) a first polypeptide antigen or antigenic fragment thereof containing 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 amino acid sequence to (a) a biotin binding site, (b) a first polypeptide antigen or antigenic fragment thereof containing at least 90%, at least 91%, at least 92%, at least 93%, or less identical amino acid sequence to (a) a biotin binding site, (b) a first polypeptide antigen or antigenic fragment thereof containing at least 90%, at least 91%, at least 92%, at least 93%, or less identical amino acid sequence to (a) a biotin binding site, (b) a first polypeptide antigen or antigenic fragment thereof containing at least 90%, at least 91%, at least 92%, at least 93%, or less identical amino acid sequence to (a) a biotin binding site, (b) a first polypeptide antigen or antigenic fragment thereof containing 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% (ii) a second polypeptide antigen or antigenic fragment thereof containing an amino acid sequence that is 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical, and (ii) a biotinylated polysaccharide antigen selected from each serotype of Streptococcus pneumoniae 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. A second multi-type immunogenic complex comprising, wherein each biotinylated polysaccharide antigen is non-covalently complexed with the biotin-binding site of the second fusion protein, wherein the second fusion protein comprises (a) a biotin-binding site and (b) SEQ ID NO: 13, with 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%, and at least 99%.(c) comprising a first polypeptide antigen or its antigenic fragment containing 5% or 100% identical amino acid sequences, and SEQ ID NO: 11 containing a second polypeptide antigen or its antigenic fragment antigen containing 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 amino acid sequences.

[0029] In some embodiments of the various aspects described herein, the biotin-binding site in the immunogenic complex fusion protein described herein is or comprises a rizavidin polypeptide. In some embodiments, the rizavidin polypeptide is (i) a polypeptide or biotin-binding fragment thereof containing 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% the same amino acid sequence as SEQ ID NO: 1, or (ii) a polypeptide or biotin-binding fragment thereof containing 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% the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 3.

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

[0031] In some embodiments of the various aspects described herein, the pharmaceutical compositions described herein further comprise one or more of the following: (a) a buffer (e.g., including, but not limited to, histidine buffer, succinate buffer, and / or 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, the pharmaceutical compositions described herein comprise either a succinate buffer or a buffer containing the succinate buffer.

[0032] Another aspect of this disclosure provides dosages of the pharmaceutical compositions described herein. In some embodiments, the amount of aluminum in the dosage is less than 0.625 mg, for example, about 0.125 mg to about 0.625 mg. In some embodiments, the amount of aluminum in the dosage is about 0.325 mg to about 0.175 mg. In some embodiments, the amount of aluminum in the dosage is about 0.25 mg. In some embodiments, the amount of aluminum in the dosage is at least 0.175 mg. In some embodiments, the amount of aluminum in the dosage is at least 0.125 mg. In some embodiments, the volume of the dosage is about 0.5 mL. In some embodiments, the dosage is for use in humans.

[0033] In some embodiments, delivery devices or containers containing the dosages of the pharmaceutical compositions described herein are also within the scope of this disclosure. For example, in one embodiment, what is provided herein is a syringe containing the dosages of the pharmaceutical compositions described herein. In other embodiments, what is provided herein is a vial containing one or more of the pharmaceutical compositions described herein or the dosages of the pharmaceutical compositions described herein.

[0034] The techniques described herein (e.g., compositions and methods) are useful for administration to a subject. For example, in some embodiments, the techniques described herein (e.g., compositions and methods) are useful for immunizing a subject. In certain embodiments, the subject suitable for the techniques described herein is a human subject. In some embodiments, the human subject is an adult human subject. In some embodiments, the human subject is a pediatric subject. For example, one aspect of the present disclosure provides a method comprising the step of administering an immunologically effective amount of the pharmaceutical composition described herein or a dosage of the pharmaceutical composition described herein to a subject. Another aspect of the present disclosure provides a method for immunizing a subject, the method comprising the step of administering an immunologically effective amount of the pharmaceutical composition described herein or a dosage of the pharmaceutical composition described herein to a subject. In some embodiments, upon administration, the pharmaceutical composition or dosage induces an immune response to (i) at least one polysaccharide antigen and / or (ii) at least one polypeptide antigen. In some embodiments, the immune response is or includes an antibody response and / or a B cell response. In some embodiments, the immune response is at a level higher than the corresponding reference level, where the reference level is the level of immune response induced in a subject after administration of the reference composition. In some embodiments, the immune response is at least 20% higher than the corresponding reference level. In some embodiments, the reference composition comprises an immunogenic composition and an aluminum-based adjuvant or an adjuvant containing the same, wherein the aluminum concentration in the reference composition is 1.25 mg / mL or higher. [Brief explanation of the drawing]

[0035] The teachings of the present invention described herein can be better understood from the following description of various exemplary embodiments when read in conjunction with the accompanying drawings. The drawings described below are for illustrative purposes only and should be understood not to be intended to limit the scope of the teachings of the present invention in any way.

[0036] [Figure 1]Figure 1 is a schematic diagram of an exemplary MAPS technology, in which a MAPS immunogenic complex comprises one or more polypeptide antigens fused to the biotin-binding protein rizavidin or its biotin-binding domain or biotin-binding fragment, and a biotinylated antigenic polysaccharide. In this diagram, each MAPS complex is formed between one or more fusion proteins and biotinylated polysaccharides by non-covalent bonding of truncated rizavidin to biotin. [Figure 2] Figure 2 shows a schematic of an exemplary CP1 fusion protein. Such an exemplary CP1 fusion protein includes a biotin-binding protein, e.g., a truncated rizavidin protein (e.g., amino acids 45-179 of the wild-type rizavidin protein), a first linker (e.g., the GGGSSS (SEQ ID NO: 38) linker), an SP1500 polypeptide (e.g., amino acids 27-278 of the full-length Streptococcus pneumoniae SP1500 polypeptide), a second linker (e.g., amino acid sequence AAA), and an SP0785 polypeptide (e.g., amino acids 33-399 of the full-length Streptococcus pneumoniae SP0785 polypeptide). In some embodiments, the CP1 fusion protein may further include a detectable or purified tag (e.g., a His tag). Amino acid sequence AAA may be derived from a NotI site on the pET21 / 24 plasmid or may be synthesized. For the GGGGSSS (SEQ ID NO: 38) linker, the SSS amino acid sequence may be derived from the SacI site on the pET21 / 24 plasmid, and the GGGG (SEQ ID NO: 67) amino acid sequence may be added to create a mobile linker with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO: 38) linker may be synthesized. [Figure 3]Figure 3 shows a schematic of an exemplary SPP2 fusion protein. Such an exemplary SPP2 fusion protein includes a biotin-binding protein, e.g., a truncated rizavidin protein (e.g., amino acids 45-179 of the wild-type rizavidin protein, denoted as Rhavi), a first linker (e.g., the GGGSSS (SEQ ID NO: 38) linker), a pneumolysin (Ply) polypeptide (e.g., amino acids 2-470 of the full-length Ply polypeptide of Streptococcus pneumoniae, including the G294P, D385N, C428G, and W433F mutations, denoted as PdT(G294P)), a second linker (e.g., the GGGSSS (SEQ ID NO: 38) linker), and an SP0435 polypeptide (e.g., amino acids 62-185 of the full-length SP0435 polypeptide of Streptococcus pneumoniae). In some embodiments, the SPP2 fusion protein may further include a detection or purification tag (e.g., a His tag). For the GGGGSSS (SEQ ID NO: 38) linker, the SSS amino acid sequence may be derived from the SacI site on the pET21 / 24 plasmid, and the GGGG (SEQ ID NO: 67) amino acid sequence may be added to create a mobile linker with minimal steric hindrance. Alternatively, the GGGGSSS (SEQ ID NO: 38) linker may be synthesized. [Figure 4] Figure 4 shows a schematic of a plasmid construct consisting of a pET-24a(+) vector containing a sequence encoding an exemplary SPP2 fusion protein. [Figure 5]Figure 5 is a flowchart illustrating exemplary upstream (top) and downstream (bottom) manufacturing processes for fusion proteins (e.g., CP1 and SPP2) as described herein. The exemplary SPP2 polypeptide is a fusion protein comprising truncated rizavidin [amino acids 45-179 of the full-length rizavidin protein], a first linker (e.g., GGGSSS (SEQ ID NO: 38) linker), pneumolysin (Ply) polypeptide, a second linker (e.g., GGGSSS (SEQ ID NO: 38) linker), and SP0435 polypeptide. The exemplary CP1 polypeptide is a fusion protein comprising truncated rizavidin [amino acids 45-179 of the full-length rizavidin protein], a first linker (e.g., GGGSSS (SEQ ID NO: 38) linker), SP1500 polypeptide, a second linker (e.g., amino acid sequence AAA), and SP0785 polypeptide. SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFF: Tangential flow filtration; DS: Active pharmaceutical ingredient [Figure 6-1] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-2] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-3] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-4]Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-5] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-6] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-7] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-8] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-9] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-10] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-11] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-12] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-13] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-14] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-15]Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-16] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-17] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-18] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-19] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-20] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-21] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-22] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-23] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 6-24] Figure 6 is a table showing exemplary structures of antigenic polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, and 35B. [Figure 7] Figure 7 is a flowchart illustrating an exemplary manufacturing process of a MAPS immunogenic complex, which includes biotinylation of an antigenic polysaccharide and assembly with a fusion protein(s) as described herein (e.g., CP1 or SPP2). [Figure 8] Figure 8 is a flowchart illustrating an exemplary manufacturing process for a MAPS vaccine. For example, such an exemplary process can be used to produce a MAPS34 vaccine containing MAPS immunogenicity complexes containing capsular polysaccharides derived from 34 different serotypes of Streptococcus pneumoniae. DMAP: 4-dimethylaminopyridine; PS: polysaccharide † In-process testing [Figure 9]Figure 9 is a schematic diagram illustrating various multivalent vaccine candidates that combine different polysaccharides (serotypes) of Streptococcus pneumoniae with antigenic fusion proteins CP1 and SPP2. The immunization groups (A, B, C, S, T) corresponding to each vaccine candidate are shown. "Prevnar 13" 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 CP1) includes the Prevnar 13 serotype and 11 new Pn-MAPS-24v serotypes other than serotype 2. Group B > MAPS34 (all on CP1) includes the Prevnar 13 serotype, 11 new Pn-MAPS-24v serotypes, and 10 new 30+ serotypes. Group C > MAPS34 (24-CP1 / 10-SPP2) includes the Prevnar 13 serotype and 11 new Pn-MAPS-24v serotypes on CP1, and 10 new 30+ serotypes on SPP2. Group S > MAPS34 (24 / 10 selected) includes Prevnar type 13 serotypes, 11 new Pn-MAPS-24v serotypes, and 10 new 30+ 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 CP1, and serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F on SPP2). Group T > MAPS34 (24 / 10 selected) includes Prevnar type 13 serotypes, 11 new serotypes for Pn-MAPS-24v, and 10 new serotypes for 30+ (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 CP1, and serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F on SPP2). [Figure 10] Figure 10 shows representative IgG levels for 34 serotypes of capsular polysaccharides in P2 rabbit serum, for each immunization group A, B, C, S, and T. The upper figure shows the results for the first 24 serotypes (also included in the applicant's previous vaccine description, see WO 2020 / 056202). The lower figure shows the results for an additional 10 serotypes. The results are expressed as IgG levels in arbitrary units (au) for the upper figure and in ng / mL for the lower figure. In the graphs, each serotype is associated with a set of five bar graphs, which show the IgG levels of the immunization group in the following order: A, B, C, S, and T. [Figure 11] Figure 11 shows the relative antibody titers against the capsular polysaccharides of the indicated serotypes, comparing baseline MAPS-34 (all on CP1) [immunization group B] with MAPS-34 (24-CP1 / 10-SPP2) [immunization group C], MAPS-34 (selected 24 / 10) [immunization group S, aluminum dose 0.0625 mg], and MAPS-34 (selected 24 / 10) [immunization group T, aluminum dose 0.025 mg]. Geometric mean titer (GMT) ratios and 95% confidence intervals are shown in the graph. For each immunization group, a star indicates that a specific capsular polysaccharide is complexed with the antigenic fusion protein SPP2. Black stars correspond to immunization group C, and gray stars correspond to immunization groups S and T. No star indicates that the capsular polysaccharide is complexed with the antigenic fusion protein CP1. In the graph, each serotype is associated with a set of three circles, and the GMT ratios of the immunized groups are shown in the following order: C, S, and T. [Figure 12] Figure 12 shows the relative antibody titers against the capsular polysaccharides of the indicated serotypes, comparing the baseline MAPS-34 (selected 24 / 10) [immunization group S, aluminum dose 0.0625 mg] with MAPS-34 (selected 24 / 10) [immunization group T, aluminum dose 0.025 mg]. Geometric mean titer (GMT) ratios and 95% confidence intervals are shown in the graph. [Figure 13]Figure 13 shows representative IgG levels of the CP1 fusion protein against the SP1500-SP0785 portion of Streptococcus pneumoniae in P0, P1, and P2 rabbit serum from each immunization group A, B, C, S, and T. Each point on the graph represents one rabbit. Results are shown on the graph in μg / ml with 95% confidence intervals and are also presented below the graph as geometric mean (μg / ml). In the graph, each immunization group is associated with a set of points in three vertical clusters, representing P0, P1, and P2, respectively. [Figure 14] Figure 14 shows representative IgG levels of the SPP2 fusion protein against the PdT(G294P) portion of Streptococcus pneumoniae in P0, P1, and P2 rabbit serum from each immunization group A, B, C, S, and T. Each point on the graph represents one rabbit. Results are shown on the graph in μg / ml with 95% confidence intervals and are also presented below the graph as geometric mean (μg / ml). In the graph, each immunization group is associated with a set of points in three vertical clusters, representing P0, P1, and P2, respectively. [Figure 15] Figure 15 shows the anti-SP1500-SP0785 IgG titers (μg / mL) in P2 rabbit serum derived from each immunization group C, S, and T, and is graphed against the anti-PdT(G294P) IgG titer from the same immunization group. [Figure 16] Figure 16 shows the representative half-limiting concentrations (IC50) of neutralizing antibodies against the hemolytic activity of native pneumolysin in P1 and P2 rabbit serum from immunization groups C, S, and T, respectively. Each point on the graph represents one rabbit. The results are presented on the graph as IC50 (serum dilution) with 95% confidence intervals, and the geometric mean (IC50) is shown in the table below the graph. [Figure 17] Figure 17 shows the anti-PdT(G294P) IgG titers (μg / mL) in P2 rabbit serum derived from each immunization group C, S, and T, and is graphed against the median inhibitory concentration (IC50) of the pneumolysin neutralizing antibody derived from the same immunization group. [Figure 18]Figure 18 is a schematic diagram illustrating various multivalent vaccine candidates that combine different polysaccharides (serotypes) and antigenic fusion proteins CP1 and SPP2 of Streptococcus pneumoniae. The immunized cohorts (MAPS-24 (all on CP1), MAPS-34 (all on CP1), and MAPS-34 (24-CP1 / 10-SPP2)) correspond to each vaccine candidate, as shown. All three vaccine candidates contained 24 polysaccharide serotypes 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 on CP1. For MAPS-34 (all on CP1) and MAPS-34 (24-CP1 / 10-SPP2), 10 additional polysaccharide serotypes 6C, 7C, 15A, 16F, 23A, 23B, 24F, 31, 35B, and 38 were each on either CP1 or SPP2. [Figure 19A] Figure 19A shows representative IgG levels for 24 capsular polysaccharides in P2 rabbit serum for each aluminum formulation group (Group A: High, Group B: Medium, and Group C: Low) for MAPS-24 (all on CP1). The results are expressed in arbitrary units (au) for IgG levels. In the graph, each serotype is associated with a set of three bar graphs, showing the IgG levels of the aluminum groups in the following order: A, B, and C. [Figure 19B] Figure 19B shows the relative antibody titers against capsular polysaccharides for the indicated serotypes in MAPS-24 (all on CP1), comparing the titers of aluminum group C (low) and group B (medium) to the baseline titer of group A (high). Geometric mean titer (GMT) ratios and 95% confidence intervals are shown on the graph. In the graph, each serotype is associated with a pair of circles, where the first circle represents the GMT ratio for group B and the second circle represents the GMT ratio for group C. [Figure 20]Figure 20 shows the representative IgG levels of CP1 fusion proteins in P1 and P2 rabbit serum derived from MAPS-24 (all on CP1) against the SP1500-SP-0785 portion of Streptococcus pneumoniae for three aluminum preparation groups (Group A: high, Group B: medium, and Group C: low). [Figure 21A] Figure 21A shows representative IgG levels for 34 capsular polysaccharides in P2 rabbit serum for each aluminum formulation group (Group A: high, Group B: medium, and Group C: low) for MAPS-34 (all on CP1). The results are expressed in arbitrary units (au) for IgG levels. In the graph, each serotype is associated with a set of three bar graphs, showing the IgG levels of the aluminum groups in the following order: A, B, and C. [Figure 21B] Figure 21B shows the relative antibody titers against capsular polysaccharides for the serotypes shown for MAPS-34 (all on CP1), where titers in aluminum group C: low and group B are compared to the baseline titer in group A: high. Geometric mean titer (GMT) ratios and 95% confidence intervals are shown on the graph. In the graph, each serotype is associated with a pair of circles, where the first circle represents the GMTR for group B and the second circle represents the GMTR for group C. [Figure 22] Figure 22 shows the representative IgG levels of CP1 fusion proteins in P1 and P2 rabbit serum derived from MAPS-34 (all on CP1) against the SP1500-SP-0785 portion of Streptococcus pneumoniae for three aluminum formulation groups (Group A: high, Group B: medium, and Group C: low). [Figure 23A] Figure 23A shows representative IgG levels for 34 capsular polysaccharides in P2 rabbit serum for each aluminum formulation group (Group A: high, Group B: medium, and Group C: low) of MAPS-34(24-CP1 / 10-SPP2). The results are expressed in arbitrary units (au) as IgG levels. In the graph, each serotype is associated with a set of three bar graphs, showing the IgG levels of the aluminum groups in the following order: A, B, and C. [Figure 23B] Figure 23B shows the relative antibody titers against capsular polysaccharides for the serotypes shown for MAPS-34 (24-CP1 / 10-SPP2), where aluminum group C (low) and group B (medium) are compared to the baseline titer of group A (high). Geometric mean titer (GMT) ratios and 95% confidence intervals are shown on the graph. In the graph, serotypes are associated with pairs of circles, where the first circle represents the GMTR for group B and the second circle represents the GMTR for group C. [Figure 24A] Figure 24A shows the representative IgG levels of the CP1 fusion protein in P1 and P2 rabbit serum derived from MAPS-34 (24-CP1 / 10-SPP2) against the SP1500-SP-0785 portion of Streptococcus pneumoniae for three aluminum preparation groups (Group A: high, Group B: medium, and Group C: low). [Figure 24B] Figure 24B shows the representative IgG levels of the SPP2 fusion protein derived from MAPS-34 (24-CP1 / 10-SPP2) in P1 and P2 rabbit serum against the PdT(G294P) portion of Streptococcus pneumoniae for three aluminum preparation groups (Group A: high, Group B: medium, and Group C: low).

[0037] Definition

[0038] In this application, unless otherwise clearly indicated by the context, (i) the term “one (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 the enumerated 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 allow for standard variations that can be understood by a person of the ordinary art, and (v) where a range is provided, it includes both endpoints.

[0039] about When the term “about” is used herein in reference to a value, it refers to a value similar to the value being referenced in that context. Generally, a person skilled in the art familiar with the context will be able to understand the degree of variation that is encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values ​​among 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than or equal to the value being referenced.

[0040] Administration As used herein, the term “administration” typically refers to administering a composition to a subject or system to achieve delivery of the active substance, or a substance contained therein, of the composition. A person of ordinary skill in the art will be able to recognize the various routes that may be used to administer a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial infusion), buccal, via the skin (e.g., one or more of the following, or including, topical, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intraarachnoid, intravenous, intraventricular, intracerebral, intrahepatic, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal infusion), vaginal, vitreous, etc. In some embodiments, administration may consist of a single dose only. In some embodiments, administration may consist of the application of a certain number of doses. In some embodiments, administration may consist of intermittent dosing (e.g., multiple doses spaced apart in time) and / or periodic dosing (e.g., individual doses spaced apart in time). In some embodiments, administration may consist of continuous dosing (e.g., perfusion) for at least a selected period.

[0041] agent In general, the term “active substance” as used herein may be used to refer to a compound or entity of any chemical classification, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Under appropriate circumstances, as may be apparent to those skilled in the art, the term may be used to refer to a cell or organism, or a fraction, extract thereof, or an entity that is or contains components thereof. Alternatively or additionally, as may be apparent to the context, the term may be used to refer to a natural product found in nature and / or obtained from nature. In some cases, as may also be apparent to the context, the term may be used to refer to one or more entities of artifacts designed, engineered, and / or produced through the work of human hands and / or not found in nature. In some embodiments, the active substance may be available in isolated or pure form, and in some embodiments, the active substance may be available in crude form. In some embodiments, potential active substances may be provided as aggregates or libraries, for example, that can be screened to identify or characterize the active active substances therein. In some cases, the term “active substance” may refer to a compound or entity that is or contains a polymer, and in some cases, the term may refer to a compound or entity that contains one or more polymer moieties. In some embodiments, the term “active substance” may refer to a compound or entity that is not a polymer, and / or a compound or entity that is substantially free of any polymer, and / or a compound or entity that is substantially free of one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or substantially does not contain any polymer moieties.

[0042] amino acid In its broadest definition, the term “amino acid” as used herein means any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the 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, and in some embodiments, an amino acid is an L-amino acid. A “standard amino acid” refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A “non-standard amino acid” refers to any amino acid other than a standard amino acid, regardless of whether it is synthesized and prepared or obtained from a natural source. In some embodiments, an amino acid (including amino acids at the carboxyl and / or amino terminus in a polypeptide) may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified compared to their general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may alter, for example, the cyclic half-life of a polypeptide containing a modified amino acid compared to one containing the same amino acid except that which is unmodified. In some embodiments, such modifications do not significantly alter the activity associated with a polypeptide containing a modified amino acid compared to one containing the same amino acid except that which is unmodified. As may be apparent from the context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid, and in some embodiments, the term “amino acid” may be used to refer to an amino acid residue of a polypeptide.

[0043] antigen As used herein, the term “antigen” means (i) an activator that induces an immune response, and / or (ii) an activator that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen induces a humoral response (e.g., including the production of antigen-specific antibodies), and in some embodiments, an antigen induces a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen induces both a humoral and a cellular response. In some embodiments, an antigen may or may not bind to an antibody and induce a specific physiological response in the body. Generally, an antigen may be or include any chemical entity such as, for example, small molecules, nucleic acids, polypeptides, carbohydrates, lipids, or polymers (in some embodiments, other than biomacromolecules (e.g., other than nucleic acids or amino acid polymers)). In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a polysaccharide. Those skilled in the art will generally recognize that antigens may be provided in isolated or pure form, or in crude form (for example, together with other materials, such as extracts such as cell extracts, or other relatively crude preparations of antigen-containing sources). In some embodiments, the antigens used in accordance with this disclosure are provided in crude form. In some embodiments, the antigen is a recombinant antigen. In some embodiments, the antigen is a polypeptide or polysaccharide that, when administered to a subject, induces a specific and / or clinically significant immune response to such polypeptide or polysaccharide. In some embodiments, the antigen is selected to induce a specific and / or clinically significant immune response to such polypeptide or polysaccharide. As will be recognized by those skilled in the art, the antigen may be the full-length or partial polypeptide antigen containing one or more B cell antigenic determinants and / or one or more T cell antigenic determinants.

[0044] associated with Where the term “associated” is used herein, two entities are associated with each other if the existence, level, and / or form of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with each other if they directly or indirectly interact with each other and are and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically interacting with each other are covalently linked. In some embodiments, two or more entities that are physically interacting with each other are not covalently linked but are non-covalently associated by means such as affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0045] Biotin binding site As used herein, the term "biotin-binding site" refers to a biotin-binding protein, its biotin-binding fragment, or its biotin-binding domain.

[0046] join As used herein, the term “binding” is to be understood to typically refer to non-covalent bonding between two or more entities. A “direct” binding involves physical contact between entities or sites, while an indirect binding involves physical interaction through physical contact with one or more intermediate entities. Bonding between two or more entities can typically be evaluated in any of the following situations, including when the interacting entities or sites are studied alone or in relation to a more complex system (e.g., covalently or otherwise interacting with a carrier entity, and / or in a biological system or cell).

[0047] Carrier protein As used herein, the term “carrier protein” refers to a protein or peptide that binds to, complexes with, or otherwise interacts with a hapten (e.g., a small peptide or lipid) or a lowly immunogenic antigen (e.g., a polysaccharide) and induces or enhances an immune response to such a bound, complexed, or otherwise interacting hapten (e.g., a small peptide or lipid) or lowly immunogenic antigen (e.g., a polysaccharide). In some embodiments, such an immune response is or includes a response to the hapten or lowly immunogenic antigen that binds to, complexes with, or otherwise interacts with such a carrier protein. In some embodiments, such an immune response is or includes a response to both the carrier protein and the hapten or lowly immunogenic antigen that binds to, complexes with, or otherwise interacts with such a carrier protein. In some embodiments, no significant immune response occurs to the carrier protein itself. In some embodiments, an immune response to the carrier protein may be detectable, but in some embodiments, the immune response to such a carrier protein is potent. In some embodiments, the carrier protein binds to, complexes with, or otherwise interacts with one or more other molecules.

[0048] Establishment As used herein, the term “establishment” generally refers to the ability of a microorganism to grow on a target site or surface. For example, the term “establishment” refers to the ability of a microorganism (e.g., bacteria) to grow on an anatomical site of a host (e.g., mucous membrane, gastrointestinal tract, injury site, organ, etc.).

[0049] Combination therapy As used herein, the term “combination therapy” refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously. In some embodiments, such regimens may be administered sequentially (e.g., all “dosages” of the first regimen are administered before any of the dosages of the second regimen). In some embodiments, such agents are administered in overlapping drug regimens. In some embodiments, “administration” of combination therapy may include administering one or more agents or modalities in combination to a subject receiving other agents or modalities. For clarity, in combination therapy, individual agents do not need to be administered together in a single composition (or not necessarily simultaneously), but in some embodiments, two or more agents or their active portions may be administered together with a combination composition, or together as part of a combination composition (e.g., as part of a single chemical complex or covalent bond).

[0050] domain As used herein, the term “domain” refers to a section or part of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of an entity such that, when the domain is physically separated from the rest of the original entity, it substantially or completely retains that particular structural and / or functional feature. Alternatively or additionally, a domain may be a part of an entity that, when separated from its (original) entity and linked to a different (recipient) entity, substantially retains and / or imparts to the recipient entity one or more structural and / or functional features characterized in the original entity. In some embodiments, a domain is a section or part of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, the domain is a section of a polypeptide, and in such embodiments, the domain is characterized by specific structural elements (e.g., specific amino acid sequences or sequence motifs, α-helix properties, β-sheet properties, coiled-coil properties, random-coil properties, etc.) and / or specific functional features (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0051] Dosage form or unit dosage form Those skilled in the art will understand that the term “dosage form” may be used to refer to a physically independent unit of an active substance (e.g., a therapeutic or diagnostic substance) administered to a subject. Typically, each such unit contains a predetermined amount of the active substance. In some embodiments, such an amount is a unit dose (or a portion thereof) appropriate for administration in accordance with a drug regimen (i.e., a therapeutic drug regimen) that has been established to correlate with a desired or beneficial outcome when administered to a relevant population. Those skilled in the art will understand that the total amount of a therapeutic composition or active substance administered to a particular subject may be determined by one or more attending physicians and may include administration in multiple dosage forms.

[0052] Medication regimen Those skilled in the art will understand that the term “medication regimen” can be used to refer to a set of (typically more than one) unit doses administered individually to a subject, typically at intervals. In some embodiments, a particular therapeutic agent has a recommended drug regimen, which may comprise one or more doses. In some embodiments, a drug regimen comprises multiple doses, each dose separated from the others by a time interval. In some embodiments, individual doses are separated from each other by equal periods of time. In some embodiments, a drug regimen comprises multiple doses, each dose separated by at least two different time intervals. In some embodiments, all doses in a drug regimen are of the same unit dose. In some embodiments, different doses in a drug regimen are of different amounts. In some embodiments, a drug regimen comprises a first dose at a first dose, followed by one or more additional doses at a second dose different from the first dose. In some embodiments, the drug regimen includes a first dose at a first dose, followed by one or more additional doses at a second dose equal to the first dose. In some embodiments, the drug regimen correlates with a desired or beneficial outcome when administered within a relevant population (i.e., it is a therapeutic drug regimen).

[0053] piece The “fragments” of a substance or entity described herein include an independent part of the whole, but have a structure that lacks one or more parts found in the whole. In some embodiments, a fragment consists of such an independent part. In some embodiments, a fragment includes an independent part of the whole, which shares one or more functional features found in the whole. In some embodiments, a fragment consists of such an independent part. In some embodiments, a fragment consists of or includes elements or parts of a characteristic structure found in the whole. In some embodiments, a polymer, for example, a polypeptide or polysaccharide fragment, 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 monomer units (e.g., residues) found throughout the polymer. In some embodiments, polymer fragments comprise or consist 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 monomer units (e.g., residues) found throughout the polymer. The whole substance or entity may, in some embodiments, be referred to as the “parent” of the whole.

[0054] homology As used herein, the term “homology” refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered “homological” to one another if their sequences are identical by 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%. In some embodiments, polymer molecules are considered “homologous” to one another when 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 (for example, containing residues with the relevant chemical properties in corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids and / or having “polar” or “nonpolar” side chains. Substituting one amino acid for another of the same type can often be considered a “homologous” substitution.

[0055] identity As used herein, the term “identity” refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymer molecules are considered “substantially identical” to one another if their sequences are identical by 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%. The calculation of the identity percentage of two nucleic acid or polypeptide sequences can be carried out, for example, by aligning the two sequences for the purpose of optimal comparison (for example, gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for the purpose of comparison). In certain embodiments, the length of the sequences to be aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Subsequently, nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence, then these molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the percentage of identity between two nucleotide sequences can be determined using the algorithm by Meyers and Miller (1989), which is incorporated into the ALIGN program (version 2.0).In some exemplary embodiments, the comparison of nucleic acid sequences generated by the ALIGN program uses a PAM120 weight residue table, a gap length penalty 12, and a gap penalty 4. Alternatively, the identity percentage between two nucleotide sequences can also be determined using the GAP program in the GCG software package, which uses the NWSgapdna.CMP matrix.

[0056] Improvement, increase, suppression, or decrease As used herein, the terms “improve,” “increase,” “suppress,” “decrease,” or their grammatical equivalents, indicate values ​​relative to a baseline or other reference measure. In some embodiments, a suitable reference measure may be or include a measurement in a particular system (e.g., in a single object) in the absence of a particular active substance or treatment (e.g., before and / or after), or under other comparable conditions in which a suitable comparable reference active substance is present. In some embodiments, a suitable reference measure may be or include a measurement in a comparable system known or expected to respond in a particular manner in the presence of a relevant active substance or treatment.

[0057] immunological effective dose or immunological effective dosage As used herein, “immunologically effective amount” or “immunologically effective dose” means an amount of antigenic or immunogenic substance, such as an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, that is sufficient to enhance the subject’s own immune response to subsequent exposure to a pathogen when administered to a subject as a single dose or as part of a series of doses. In some embodiments, the pathogen is Streptococcus pneumoniae. In some embodiments, the immune response is against one or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against two or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against nine or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against thirteen or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against fifteen or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against twenty-three or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against 24 or more different serotypes of Streptococcus pneumoniae. The immunological effective dose may vary depending on the subject to be treated, the species of the subject, and the degree of immune response to be induced. In some embodiments, the immunological effective dose is sufficient to treat or protect a subject that has or is at risk of having the disease. In some embodiments, the immunological effective dose is a non-toxic but sufficient 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, the immunological effective dose is sufficient to induce an immune-protective response when administered to a subject.

[0058] Immune defense response or defense response As used herein, “immunoprotective response” or “protective response” refers to an immune response mediated by immunological memory induced by an antigen or immunogen. In some embodiments, an immunoprotective response is induced by administering a substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition to a subject. In some embodiments, immunoprotection includes one or more of the following: active immune surveillance, a more rapid and effective response upon immune activation compared to the response observed in an untreated subject, efficient clearance of an activator or pathogen, and subsequent rapid resolution of inflammation. In some embodiments, the immunoprotective response is an adaptive immune response. In some embodiments, the immunoprotective response is sufficient to protect an immunized subject from a proliferative infection (e.g., Streptococcus pneumoniae infection) by a specific pathogen or group of pathogens targeted by a vaccine.

[0059] immunization As used herein, “immunization” or its grammatical equivalent means the process of inducing an immune response in a subject to an infectious organism or agent (“active immunization”), or providing a subject with an immune system component against an infectious organism or agent (“passive immunization”). In some embodiments, immunization includes administering one or more antigens, immunogens, immunogenic complexes, vaccines, immune molecules, such as antibodies, immune serum, immune cells such as T cells or B cells, or pharmaceutical compositions to a subject. In some embodiments, immunization is carried out by administering an immunologically effective amount of a substance, such as an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, immune molecule, such as antibodies, immune serum, immune cells such as T cells or B cells, or pharmaceutical compositions to a subject. In some embodiments, immunization results in an immune-protective response in a subject. In some embodiments, active immunization is carried out by administering an antigenic or immunogenic substance, such as an antigen, immunogen, immunogenic complex, vaccine, or pharmaceutical composition to a subject. In some embodiments, passive immunization is carried out by administering immune system components, such as immune molecules, such as antibodies, immune serum, or immune cells such as T cells or B cells, to the target.

[0060] Isolated As used herein, the term “isolated,” or its grammatical equivalent, means (1) separated from at least some of the components with which they were initially interacting (whether in nature and / or in an experimental setting), and / or (2) substances and / or entities designed, produced, prepared, and / or manufactured by human hands. 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 interacting. In some embodiments, the isolated active ingredient is pure by 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%. 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 be considered “isolated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), in which case the isolation rate or purity of the substance is calculated without including such carriers or excipients. To give just one example, in some embodiments, a naturally occurring biopolymer such as a polypeptide or polysaccharide is considered “isolated” if, for reasons of its origin or source, it does not interact with some or all of the components that naturally accompany it in nature; b) it substantially does not contain other polypeptides or nucleic acids of the same kind from the species that naturally produces it; or c) it is expressed by cells or other expression systems that are not of the species that naturally produce it, or is otherwise related to its components. Accordingly, for example, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or synthesized in a cell system different from the one that naturally produces it is considered an “isolated” polypeptide or polysaccharide.Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide subjected to one or more purification techniques may be considered an “isolated” polypeptide or polysaccharide insofar as it has been separated from a) other components with which it interacts in nature, and / or b) other components with which it interacted when it was first produced.

[0061] 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, a person skilled in the art will understand that a polypeptide having two or more functional or organizational domains in its structure often contains a sequence of amino acids between them that link such domains together. In some embodiments, a polypeptide containing a linker component has the general form S1-L-S2 overall structure, where S1 and S2 represent two domains that may be the same or different and are associated with each other by a linker (L). In some embodiments, the polypeptide linker has an amino acid length of 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. In some embodiments, the linker tends not to adopt a rigid three-dimensional structure, but rather is characterized by imparting mobility (flexibility) to the polypeptide. Various different linker components that can be appropriately used in the engineering operations of polypeptides (e.g., fusion polypeptides) are known in the art (Holliger et al., 1993; Poljak, 1994).

[0062] mutation As used herein, the term “mutation” is used to refer to a difference in the sequence of a nucleic acid or polypeptide compared to a reference sequence. Exemplary types of mutations include, but are not limited to, insertions, deletions, and substitutions. To avoid ambiguity, the term “mutation” is understood by those skilled in the art to include a difference compared to a reference, and does not necessarily refer to or imply a change occurring within any of the specific sequences of interest.

[0063] Non-inferiority As used herein, the term “non-inferiority” in the context of evaluating test drug compositions means that the test drug composition is at least as effective as the reference composition (for example, in terms of immunogenicity and / or functional antibody titer produced by the test composition). In some embodiments, non-inferiority is demonstrated if the lower limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the test drug composition to the reference composition is at least greater than or equal to 0.4, or greater than (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 greater than). For example, in some embodiments, the immunogenic compositions (e.g., vaccines) described herein are non-inferior to a reference vaccine (e.g., PCV13, PCV20, or PPSV23) if the lower limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to the reference vaccine is greater than or equal to 0.4, or greater than (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 greater than this). In some embodiments, the immunogenic compositions (e.g., vaccines) described herein are non-inferior to a reference vaccine (e.g., PCV13, PCV20, or PPSV23) if the lower limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to the reference vaccine is greater than or equal to 0.95, or greater than (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 greater than this).In some embodiments, the immunogenic compositions (e.g., vaccines) described herein are non-inferior to a reference vaccine (e.g., PCV13, PCV20, or PPSV23) if the serum conversion rate, or the proportion of vaccine recipients showing an immune response, exceeds a predetermined threshold, for example, if the lower limit of the 95% confidence interval for the difference in the proportion of subjects undergoing serum conversion after immunization with the immunogenic compositions (e.g., vaccines) described herein, or after immunization with a reference vaccine (e.g., PCV13, PCV20, or PPSV23), is greater than -0.10.

[0064] Pharmaceutical composition As used herein, the term “pharmaceutical composition” refers to a composition in which an active ingredient is formulated with one or more pharmaceutically acceptable excipients and / or carriers. In some embodiments, the active ingredient is present in a unit dose appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including, but are not limited to, oral administration, e.g., liquid drugs (aqueous or nonaqueous solutions or suspensions), tablets, e.g., for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as sterile solutions or suspensions, or as sustained-release formulations, topical application, e.g., as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or oral cavity, vaginal or rectal administration, e.g., as pessaries, creams, or foams, sublingual, ocular, transdermal, or nasal, intrapulmonary, and administration to other mucosal surfaces.

[0065] Pharmaceutically acceptable As used herein, the term “pharmaceutically acceptable” applied to carriers, diluents, or excipients used to formulate the compositions disclosed herein means that the carrier, diluent, or excipient must be compatible with the other components of the composition and must not be harmful to its recipient.

[0066] polysaccharide As used herein, the term "polysaccharide" refers to a polymeric carbohydrate molecule composed of long chains in which monosaccharide units are linked by glycosidic bonds, phosphate diester bonds, or other linkages, and which, upon hydrolysis, become constituent monosaccharides or oligosaccharides. Polysaccharides can take on a variety of structures, from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, structural polysaccharides such as cellulose and chitin, and microbial polysaccharides, as well as antigenic polysaccharides found in microorganisms, including but not limited to capsular polysaccharides (CPS), O-polysaccharides (OPS), core O-polysaccharides (COPS), and lipopolysaccharides (LPS).

[0067] polypeptide As used herein, the term “polypeptide” generally has the meaning recognized in the art of being a polymer of at least three amino acids linked together, for example, by peptide bonds. Those skilled in the art will understand that the term “polypeptide” is intended to be sufficiently general to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments retaining at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally tolerate some substitutions without impairing activity. Therefore, any polypeptide that retains activity and shares at least about 30–40%, often about 50%, 60%, 70%, or more than 80%, overall sequence identity with another polypeptide of the same classification, and further typically includes at least one region having significantly higher identity, often more than 90%, or even 95%, 96%, 97%, 98%, or 99%, in one or more highly conserved regions, usually comprising at least 3–4 and up to 20 or more amino acids. A polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, the protein may contain native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof.

[0068] prevention When used herein in relation to a disease, disorder, and / or medical condition, the terms “prevent” or “prevention” mean a reduction in the risk of developing a disease, disorder, and / or condition, and / or a delay in its onset, and / or a reduction in the frequency and / or severity of one or more characteristics or symptoms of a particular disease, disorder, or condition. In some embodiments, the evaluation of prevention is performed on a population basis, such that a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of a disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition, and the active ingredient is considered to “prevent” a particular disease, disorder, or condition. In some embodiments, prevention may be considered complete when the onset of a disease, disorder, or condition is delayed over a predetermined period of time.

[0069] protein As used herein, the term “protein” encompasses polypeptides. Proteins may include portions other than amino acids (e.g., glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those skilled in the art will understand that a “protein” may be a complete polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those skilled in the art will understand that a protein may include more than one polypeptide chain linked, for example, by one or more disulfide bonds, or interacting by other means. Polypeptides may include L-amino acids, D-amino acids, or both, and may include any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to mean polypeptides 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, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0070] Recombinant As used herein, the term “recombinant” is intended to mean polypeptides designed, engineered, prepared, expressed, fabricated, manufactured, and / or isolated by recombinant means; for example, polypeptides expressed using recombinant expression vectors transfected into host cells; polypeptides isolated from recombinant combinatorial human polypeptide libraries; polypeptides isolated from animals (e.g., mice, rabbits, sheep, fish, etc.) that are transgenic with respect to one or more components, parts, elements, or domains thereof, and / or genes or gene components that are directed to express them, or that are otherwise engineered to express them; and / or polypeptides prepared, expressed, fabricated, or isolated by any other means, including splicing of selected nucleic acid sequence elements or ligation of selected nucleic acid sequence elements, chemical synthesis of selected sequence elements, and / or alternative production of polypeptides or nucleic acids that encode one or more components, parts, elements, or domains thereof, and / or that are directed to express them. In some embodiments, one or more of these selected sequence elements are found in nature. In some embodiments, one or more of these selected sequence elements are designed in silico. In some embodiments, one or more of these selected sequence elements are obtained from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources, such as the germline of the source organism of interest (e.g., human, mouse, etc.).

[0071] reference As used herein, the term “reference” refers to a standard or control for the subject being compared. For example, in some embodiments, the active substance, animal, subject, population, sample, sequence, or value of interest is compared to the active substance, animal, subject, population, sample, sequence, or value of a reference or control. In some embodiments, the reference or control is tested and / or determined substantially concurrently with the test or determination of interest. In some embodiments, the reference or control is a past reference or control embodied in a tangible medium, at the discretion of the art. Typically, as will be understood by those skilled in the art, the reference or control is determined or analyzed under conditions or circumstances comparable to those being evaluated. Those skilled in the art may understand that there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control.

[0072] response As used herein, “response” to a treatment may mean any beneficial change in the subject’s condition that occurs as a result of or correlates with the treatment. Such changes may include stabilization of the condition (e.g., prevention of exacerbation that would have occurred without treatment), alleviation of the symptoms of the condition, and / or improvement in the likelihood of healing. This may also mean the response of the subject or the tumor. The response of the subject or the tumor may be measured according to a wide range of criteria, including clinical and objective criteria. Techniques for evaluating the response include, but are not limited to, clinical tests, positron emission tomography, chest X-ray CT scans, MRI, ultrasound, endoscopy, laparoscopy, the presence or level of biomarkers in samples obtained from the subject, cytological and / or histological examinations. Appropriate response criteria may be selected in any suitable form, as long as, when comparing the subject group and / or the tumor group, the group being compared is evaluated based on the same or comparable criteria for determining the response rate. A person skilled in the art will be able to select appropriate criteria.

[0073] risk As understood from the context, the “risk” of a disease, disorder, and / or condition means the likelihood that a particular subject will develop that disease, disorder, and / or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90-100%. In some embodiments, the risk is expressed as a relative risk to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or reference sample group is derived from subjects comparable to a particular subject. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher.

[0074] serotype As used herein, the term "serotype," also referred to as "serovar," means a distinct difference within a bacterial or viral species or between immune cells of different targets. These microorganisms, viruses, or cells are classified together based on their cell surface antigens, making it possible to epidemiologically classify organisms down to the subspecies level. A group of serotypes sharing common antigens may be called a serogroup, or sometimes a serocomplex.

[0075] seed As used herein, the term “species” refers to distinct immunogenic complexes comprising (i) a biotinylated polysaccharide antigen and (ii) a fusion protein comprising a biotin-binding site and one or more Streptococcus pneumoniae polypeptide antigens. In some embodiments, distinct species may differ in one or more of the fusion proteins comprising (i) a biotinylated polysaccharide antigen and (ii) a biotin-binding site and one or more Streptococcus pneumoniae polypeptide antigens.

[0076] subject As used herein, the term “Subject” means a living organism, typically a mammal (e.g., a human, and in some embodiments, a prenatal human form). In some embodiments, the Subject suffers from the relevant disease, disorder, or condition. In some embodiments, the Subject is susceptible to the disease, disorder, or condition. In some embodiments, the Subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the Subject exhibits no symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the Subject is a person who has one or more characteristics characteristic of susceptibility to or risk of the disease, disorder, or condition. In some embodiments, the Subject is a patient. In some embodiments, the Subject is a Subject who has been and / or has been given a diagnosis and / or therapy.

[0077] Superiority As used herein, the term “superiority” in the context of evaluating a test pharmaceutical composition refers to a test pharmaceutical composition that performs better than a reference composition (for example, in terms of immunogenicity and / or functional antibody titer produced by the test composition). In some embodiments, superiority is demonstrated when the upper limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the test pharmaceutical composition to the reference composition is at least 1.3 (including, for example, 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, the immunogenic composition (e.g., vaccine) described herein is superior to the reference vaccine (e.g., PCV13, PCV20, or PPSV23) if the upper limit of the 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to the reference vaccine is at least 1.3 (including, for example, 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, the immunogenic composition (e.g., vaccine) described herein is superior to the reference vaccine (e.g., PCV13, PCV20, or PPSV23) if the two-sided 95% confidence interval (CI) of the geometric mean titer (GMT) ratio of the immunogenic composition (e.g., vaccine) to the reference vaccine excludes zero.

[0078] Sensitive Subjects who are “suspected” of a disease, disorder, or condition are at risk of developing that disease, disorder, or condition. In some embodiments, subjects who are susceptible to a disease, disorder, or condition do not exhibit any symptoms of that disease, disorder, or condition. In some embodiments, subjects who are susceptible to a disease, disorder, or condition have not been diagnosed with that disease, disorder, and / or condition. In some embodiments, subjects who are susceptible to a disease, disorder, or condition are subjects who have been exposed to conditions associated with the development of that disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of a subject with a disease, disorder, or condition).

[0079] Symptoms are reduced. As used herein, “symptom reduction” means that one or more symptoms of a particular disease, disorder, or condition are reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency to a level that is statistically and / or clinically significant or meaningful. For clarity, a delay in the onset of a particular symptom is considered a form of reduction in the frequency of that symptom.

[0080] treatment As used herein, the term “treatment” (and also “to treat” or “to treat”) means any administration of therapy that partially or completely reduces, alleviates, soothes, suppresses, delays the onset of, reduces the severity of, and / or reduces the frequency of, one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of subjects that do not exhibit signs of the disease, disorder, and / or condition in question, and / or subjects that exhibit only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of subjects exhibiting one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be treatment of subjects that have been diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be treatment of subjects known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, and / or condition in question.

[0081] Vaccination As used herein, the term “vaccination” means the administration of a composition intended to induce an immune response to a pathogenic agent, for example. For the purposes of this disclosure, vaccination may be administered before, during, and / or after exposure to a pathogenic agent, and in certain embodiments, it may be administered before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination involves multiple administrations of a vaccine composition at appropriate intervals. In some embodiments, vaccination initiates immunization.

[0082] variant When used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that exhibits significant structural identity with a reference molecule but is structurally different from the reference molecule, for example, in the presence or absence of one or more chemical moieties, or at some level, compared to the reference entity. In some embodiments, a variant may also be functionally different from its reference molecule. Generally, whether a particular molecule is appropriately considered a “variant” of a reference molecule depends on the degree of structural identity with that reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. 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 the sequence of amino acids or nucleotides, and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits overall sequence identity with the reference polypeptide or nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits one or more reduced levels of biological activity compared to the reference polypeptide or nucleic acid.In some embodiments, a polypeptide or nucleic acid of interest is considered a “variant” of a reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference but has a small number of sequence modifications at specific positions. Typically, about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or less than 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 substituted residues compared to the reference. Often, the variant polypeptide or nucleic acid contains a very small number (e.g., less than 5, 4, 3, 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in specific biological activity) compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains about 5, about 4, about 3, about 2, or less than 1 addition or deletion compared to the reference, and in some embodiments, it does not contain any additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains 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, less than 6 additions or deletions compared to the reference, and generally about 5, about 4, about 3, or less than 2 additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains 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, less than 6 modifications (e.g., substitutions, additions, or deletions) in the N-terminal portion compared to the reference, and generally about 5, about 4, about 3, or less than 2 modifications. In some embodiments, the variant polypeptide or nucleic acid contains, compared to the reference, 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, less than about 6 modifications (e.g., substitutions, additions, or deletions) in the C-terminal portion. In some embodiments, the reference polypeptide or nucleic acid is naturally occurring. [Modes for carrying out the invention]

[0083] Detailed description of specific embodiments This disclosure generally relates to compositions, systems, and methods useful for providing immunogenic compositions (e.g., vaccines). In some embodiments, the insights and techniques provided herein may be useful for providing immunogenic compositions (e.g., vaccines) whose immunogenicity is enhanced to prevent a particular disease or disorder. In certain embodiments, the insights and techniques described herein are useful for MAPS-based immunogenic compositions (e.g., vaccines).

[0084] The applicant has previously used MAPS technology to develop polyvalent vaccines in which at least one polysaccharide antigen is non-covalently bound to one or more polypeptide antigens, such as polyvalent pneumococcal vaccines as described in WO 2020 / 056202 and PCT / US2022 / 043156 (for the purposes described herein, the relevant content is incorporated herein by reference in its entirety).

[0085] In particular, this disclosure provides insight that the immunogenicity of MAPS immunogenic compositions can be improved by altering the type and / or concentration of certain excipients (including, but not limited to, adjuvants) and / or formulation buffers used in the MAPS immunogenic compositions. For example, in one embodiment, aluminum-based adjuvants are widely used in vaccines to enhance their ability to elicit an immune response to the vaccine and to improve the overall efficacy of the vaccine, but this disclosure provides insight that, in particular with respect to MAPS immunogenic compositions, it may be desirable to reduce the content of aluminum-based adjuvants. Without wishing to adhere to any particular theory, the reduction in aluminum-based adjuvant content may, in some embodiments, be characterized by the amount of aluminum present in the MAPS immunogenic composition, while in some embodiments it may be characterized by the ratio of aluminum content to polysaccharide antigen content and / or polypeptide antigen content. In certain embodiments, the disclosure demonstrates that MAPS immunogenic compositions containing an aluminum-based adjuvant at an aluminum concentration of less than 1.25 mg / mL in the composition may be particularly useful in increasing the immune response to polysaccharide antigens and / or polypeptide antigens (for example, in some embodiments, B-cell immune responses and / or T-cell immune responses).

[0086] In particular, while we do not wish to be bound by any specific theory, this disclosure offers the insight that the presence of free MAPS complexes (i.e., MAPS complexes not associated with aluminum-based adjuvants) may be desirable in inducing an immune response to an antigen compared to MAPS complexes associated with aluminum-based adjuvants.

[0087] In some embodiments, this disclosure provides insight, among other things, that reducing the content of aluminum-based adjuvants may be useful in increasing the immunogenicity of MAPS immunogenic compositions (e.g., vaccines) when administered to specific patient subjects, e.g., in certain embodiments, to adult subjects. In some embodiments, such an immune response may be a B-cell immune response (e.g., to one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response may be a T-cell immune response (e.g., to one or more polysaccharide antigens and / or polypeptide antigens). In some embodiments, such an immune response may be a B-cell immune response and a T-cell immune response (e.g., to one or more polysaccharide antigens and / or polypeptide antigens).

[0088] In some embodiments, the disclosure recognizes that certain formulation buffers may be more desirable, particularly for the use of MAPS immunogenic compositions (e.g., vaccines). In some embodiments, succinate buffers may be desired for the use of such MAPS immunogenic compositions.

[0089] Accordingly, this disclosure provides, in particular, technologies (e.g., compositions, methods, and / or kits) for inducing an immune response in subjects requiring them. In certain embodiments, such technologies are useful for MAPS immunogenic compositions.

[0090] immunogenic complex This disclosure encompasses immunogenic complexes comprising one or more polypeptides and one or more polymers.

[0091] In some embodiments, the immunogenic conjugate is or is based on MAPS technology. Embodiments of MAPS technology have been previously described in WO2012 / 155007 and WO2020 / 056202 (the whole contents of which are incorporated herein by reference) and are schematically represented in Figure 1. See also Zhang et al., 2013.

[0092] In some embodiments, the immunogenic complex comprises one or more polypeptides (e.g., antigenic polypeptides) described herein, which are non-covalently complexed with one or more polymers (e.g., polysaccharides) described herein. In some embodiments, one or more polypeptides (e.g., antigenic polypeptides) are complexed with one or more polymers (e.g., polysaccharides) via affinity interactions. In some embodiments, the immunogenic complex of the present disclosure comprises one or more polypeptides (e.g., antigenic polypeptides) which are non-covalently complexed with one or more polymers (e.g., 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"). When the first affinity molecule and the complementary affinity molecule interact, one or more polypeptides (e.g., antigenic polypeptides) are non-covalently complexed with one or more polymers (e.g., antigenic polysaccharides).

[0093] In some embodiments, the immunogenic complex comprises (i) one or more polymers (e.g., antigenic polysaccharides) interacting with a first affinity molecule described herein (e.g., by chemical bonding in some embodiments), and (ii) a fusion protein comprising a complementary affinity molecule and one or more polypeptides (e.g., antigenic polypeptides) described herein. In some embodiments, the immunogenic complex comprises (i) one or more polymers (e.g., antigenic polysaccharides) interacting with a plurality of first affinity molecules described herein (e.g., by chemical bonding in some embodiments), and (ii) a fusion protein comprising a complementary affinity molecule and one or more polypeptides (e.g., antigenic polypeptides) described herein.

[0094] In some embodiments, one or more polypeptides (e.g., antigenic polypeptides) are complexed with one polymer (e.g., antigenic polysaccharide) via affinity interactions. In some embodiments, the immunogenic complex of the Disclosure comprises one or more polypeptides (e.g., antigenic polypeptides) that are non-covalently complexed with one polymer (e.g., antigenic polysaccharide) using one affinity molecule / complementary affinity molecule pair. In some embodiments, each of the affinity molecule / complementary affinity molecule pairs is identical, for example, a biotin / biotin-binding site pair. In some embodiments, the immunogenic complex comprises (i) a polymer (e.g., an antigenic polysaccharide) interacting with a first affinity molecule described herein (e.g., by chemical bonding in some embodiments), and (ii) a fusion protein comprising a complementary affinity molecule and one or more polypeptides (e.g., antigenic polypeptides) described herein. In some embodiments, the immunogenic complex comprises (i) a polymer (e.g., an antigenic polysaccharide) interacting with a plurality of first affinity molecules described herein (e.g., by chemical bonding in some embodiments), and (ii) a fusion protein comprising a complementary affinity molecule and one or more polypeptides (e.g., antigenic polypeptides) described herein. When the first affinity molecule and the complementary affinity molecule interact, one or more polypeptides (e.g., antigenic polypeptides) complex non-covalently with the polymer (e.g., antigenic polysaccharide).

[0095] In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of the following: biotin / biotin binding site, 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.

[0096] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is, for example, a biotin-binding protein, or a site therefor such as its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is rizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrAvidin, captoavidin (trademark), or their biotin-binding domain or biotin-binding fragment, or a combination thereof. In some embodiments, the biotin-binding site is a dimer, for example, a non-covalent dimer. In some embodiments, the biotin-binding site is rizavidin, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes the polypeptide of SEQ ID NO: 1, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes a polypeptide containing an amino acid sequence identical to SEQ ID NO: 1 by at least 80% (e.g., at least 90%, at least 95%, at least 98%, at least 99%, and 100%), or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes a polypeptide containing an amino acid sequence identical to SEQ ID NO: 2 or SEQ ID NO: 3 by at least 80% (e.g., at least 90%, at least 95%, at least 98%, at least 99%, and 100%), or its biotin-binding domain or biotin-binding fragment.

[0097] In some embodiments, 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 Streptococcus 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 serotypes or strains of pathogens 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. In some embodiments, one or more antigenic polysaccharides are or are derived from more than 25 serotypes or strains of the pathogen, for example, serotypes or strains 36, 37, 38, 39, 40, 45, 50, 55, or 60. In some embodiments, one or more antigenic polysaccharides are or are derived from more than 35 serotypes or strains of the pathogen, for example, serotypes or strains 36, 37, 38, 39, 40, 45, 50, 55, or 60. In some embodiments, one or more antigenic polysaccharides are or are derived from more than 70, 80, 90, or 100 serotypes or strains of the pathogen.

[0098] In some embodiments, one or more antigenic polysaccharides include one or more affinity molecules conjugated to the antigenic polysaccharide. In some embodiments, one or more affinity molecules include biotin or a derivative of biotin.

[0099] In some embodiments, the antigenic polysaccharide comprises multiple affinity molecules conjugated to the antigenic polysaccharide. In some embodiments, the affinity molecules include biotin or a derivative of biotin.

[0100] In some embodiments, one or more antigenic polypeptides are covalently linked (e.g., fused) with complementary affinity molecules as described herein. In some embodiments, the fusion protein comprises one or more antigenic polypeptides and complementary affinity molecules as disclosed herein. In some embodiments, the complementary affinity molecule is or comprises a biotin-binding site. In some embodiments, the biotin-binding site is a dimer, for example, a non-covalent dimer. In some embodiments, the biotin-binding site comprises rizavidin or its biotin-binding portion.

[0101] In some embodiments, antigenic polysaccharides and / or antigenic polypeptides that may be included in the immunogenic complex are generated recombinantly or synthetically. In some embodiments, antigenic polysaccharides and / or antigenic polypeptides that may be included in the immunogenic complex are isolated from and / or derived from natural sources. In some embodiments, antigenic polysaccharides and / or antigenic polypeptides that may be included in the immunogenic complex are isolated from bacterial cells. Exemplary polysaccharides and / or polypeptides are described below.

[0102] In some embodiments, the immunogenic conjugates of this disclosure are immunogenic conjugates such as those described in WO 2018 / 237221, WO 2020 / 056202, PCT / US2022 / 043156, or PCT / US2022 / 042964. For the purposes described herein, the contents of each of the aforementioned references are incorporated in their entirety by reference.

[0103] Antigenic polypeptide In some embodiments, the immunogenic complex described herein comprises one or more polypeptide antigens. In some embodiments, the 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, the polypeptide antigen is or comprises a polypeptide antigen associated with a pathogen (e.g., bacteria, fungi, parasites, or viruses). In some embodiments, the polypeptide antigen is or comprises a polypeptide antigen associated with a tumor.

[0104] In some embodiments, one or more polypeptide antigens contained in the immunogenic complex described herein include two or more polypeptide antigens of the same source, for example, in some embodiments, those associated with the same pathogen (e.g., bacteria, fungi, parasites, viruses, etc.) or those associated with the same cancer or tumor. In some embodiments, one or more polypeptide antigens contained in the composition described herein include polypeptide antigens associated with different pathogens (e.g., bacteria, fungi, parasites, viruses, etc.) or different cancers or tumors. For example, one or more polypeptide antigens contained in the composition described herein include (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.

[0105] In some embodiments, the polypeptide antigen is or includes a polypeptide antigen of a virus. In some embodiments, the polypeptide antigen is or includes 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 a combination thereof. In some embodiments, the polypeptide antigen is a polypeptide antigen of a coronavirus or a polypeptide antigen derived therefrom. In some embodiments, the polypeptide antigen is a polypeptide antigen of SARS-CoV-2 or a polypeptide antigen derived therefrom. In some embodiments, the polypeptide antigen is the coronavirus spike (S) protein or its antigenic fragment, the envelope (E) protein or its antigenic fragment, the membrane (M) protein or its antigenic fragment, and / or the nucleocapsid (N) protein or its antigenic fragment.

[0106] In some embodiments, the polypeptide antigen is or comprises a fungal polypeptide antigen. In some embodiments, the fungal polypeptide antigen is an aspergillosis antigen, a blastomycosis antigen, a candidiasis antigen, a chromoblastomycosis antigen, a coccidioidomycosis antigen, an emergomycosis antigen, an ophthalmic mycosis antigen, an onychomycosis antigen, a fusariosis antigen, a histoplasmosis antigen, a mucormycosis antigen, a mycetoma antigen, a paracoccidioidomycosis antigen, or a Pneumocystis pneumonia antigen. Pneumonia antigen, ringworm antigen, scedosporiosis antigen, sporotrichosis antigen, talaromycosis antigen, or a combination thereof, or including these.In some embodiments, polypeptide antigens include Aspergillus antigen, Blastomyces antigen, Candida (e.g., C. auris) antigen, Coccidioides antigen, Cryptococcus antigen (e.g., C. neoformans and C. gattii), Epidermophyton antigen, Fusarium antigen, Histoplasma antigen, Lomentospora antigen, Mazurella antigen, Mucormycetes antigen, Microsporum antigen, Paracoccidioidomycosis antigen, Pneumocystis (e.g., P. irovecii). The antigens are jirovecii antigen, Scedosporium antigen, Sporothrix antigen, Talaromyces antigen, Trichophyton antigen, or combinations thereof, or derived therefrom.

[0107] In some embodiments, the polypeptide antigen is or includes a polypeptide antigen of a parasitic protist. In some embodiments, the polypeptide antigen of a parasitic protist is or includes an amoebiasis antigen, a babesiosis antigen, a blastocystis antigen, a Chagas disease antigen, a cryptosporidiosis antigen, a giardiasis antigen, a lambliasis antigen, a malaria antigen, a toxoplasmosis antigen, a trichomoniasis antigen, or a combination thereof. In some embodiments, the polypeptide antigen is or is derived from Babesia (e.g., B. microti) antigen, Balantidium antigen, Cryptosporidium antigen, Entamoeba antigen, Giardia (e.g., G. lamblia) antigen, Leishmania antigen, Plasmodium (e.g., Plasmodium falciparum) antigen, Trichomoniasis (e.g., T. vaginalis) antigen, Trypanosoma (e.g., T. cruzi and T. brucei) antigen, or a combination thereof.

[0108] In some embodiments, the polypeptide antigen is or includes a tumor antigen. In some embodiments, the tumor antigen is a carcinoembryonic antigen (CEA), cancer / testicular antigen (e.g., New York esophageal squamous cell carcinoma 1 (NY-ESO1), mucin-1 (MUC1), and sialyl Tn (STn)), ganglioside (e.g., GM3 and GD2), p53, HER2 / neu, EGFR (e.g., EGFRvIII), melanocyte / melanoma differentiation antigen (e.g., tyrosinase, MART1, gp100, melanoma antigen (MAGE) family proteins, prostate-specific antigen (PSA), leukemia-associated antigen (LAA) (e.g., BCR-ABL, Wilms tumor protein and proteinase 3), idiotype (Id) antibody, or a combination thereof, or derived therefrom (e.g., Mitchell, Curr. Opinion. Investig. Drugs 150 (2002), Dao & Scheinberg, Best Pract. Res. Clin.). See Haematol.391 (2008). In some embodiments, the tumor antigen is or includes a tumor antigen derived from a solid tumor. In some embodiments, the tumor antigen is or includes a tumor antigen derived from a hematological malignancy. In some embodiments, the tumor antigen is or includes a tumor antigen associated with cancer. Examples of cancers known in the art include, for example, hematopoietic cancers (including leukemia, (Hodgkin and non-Hodgkin) lymphoma, myeloma, and myeloproliferative disorders), sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, throat, pharynx, and lung, liver cancer, genitourinary cancers, such as cancers of the prostate, cervix, bladder, uterus, ovaries, and uterine body, as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, melanoma of the skin or eye, endocrine cancers, thyroid cancer, parathyroid cancer, head and neck cancers, breast cancer, gastrointestinal cancers, and nervous system cancers, benign lesions, such as papillomas.

[0109] In some embodiments, the polypeptide antigen is or comprises a bacterial polypeptide antigen. In some embodiments, the bacterial polypeptide antigen is or derived from a polypeptide antigen of a Gram-negative or Gram-positive bacterium. In some embodiments, the bacterial polypeptide antigen is or comprises pneumococcus (e.g., group A, group B, and viridans) antigen, tuberculosis antigen, anthrax antigen, pertussis antigen, staphylococcus (e.g., Staphylococcus aureus) antigen, meningococcal antigen, Haemophilus antigen, Shigella antigen, Salmonella antigen, Pseudomonas antigen, Klebsiella antigen, Escherichia coli antigen, or a combination thereof. In some embodiments, the bacterial polypeptide antigen is or derived from a polypeptide antigen of Streptococcus pneumoniae.

[0110] In some embodiments, the polypeptide antigen is a polypeptide antigen of Streptococcus pneumoniae or a polypeptide antigen derived therefrom. In some embodiments, one or more polypeptide antigens are polypeptide antigens of pathogens other than Streptococcus pneumoniae or polypeptide antigens derived therefrom. In some embodiments, one or more polypeptide antigens include (i) a polypeptide antigen of Streptococcus pneumoniae or a polypeptide antigen derived therefrom, and (ii) a polypeptide antigen of pathogens other than Streptococcus pneumoniae or a polypeptide antigen derived therefrom. In some embodiments, the immunogenic complex includes one or more antigenic polypeptides or portions thereof of Streptococcus pneumoniae.

[0111] In some embodiments, the immunogenic complex described herein comprises one or more of the following antigenic polypeptides or portions thereof.

[0112] Exemplary pneumococcal polypeptide antigen: pneumocysin polypeptide In some embodiments, the antigenic polypeptide described herein is or includes a pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or includes a pneumolysin polypeptide antigen. Pneumolysin (Ply) is a protein toxin of Streptococcus pneumoniae. Ply is a cholesterol-dependent toxin of the thiol-activated cytolysin family. In some embodiments, the Ply polypeptide antigen is or includes the full-length Ply polypeptide. For example, in some embodiments, the full-length Ply polypeptide has 470 amino acids (53 kDa) and is represented by the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the Ply polypeptide antigen includes a portion of the Ply polypeptide (for example, the portion of the Ply polypeptide of SEQ ID NO: 4, which 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 consecutive amino acids of SEQ ID NO: 4). In some embodiments, the Ply polypeptide portion corresponds to a protein having amino acids 2-470 of the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the Ply polypeptide antigen contains one or more amino acid changes (e.g., deletions, substitutions, and / or insertions) from the naturally occurring wild-type Ply polypeptide sequence. For example, a Ply polypeptide antigen may contain at least 60% (for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of an amino acid sequence identical to SEQ ID NO: 4 or a portion thereof (for example, 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 represented in SEQ ID NO: 4).Alternatively, the Ply polypeptide antigen may contain a portion of the sequence (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) that is identical to SEQ ID NO: 4 by at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or more). In some embodiments, the nucleotide sequence encoding the Ply polypeptide is provided herein as SEQ ID NO: 14.

[0113] Pneumolysin is an exotoxin produced by bacteria that can induce hemolytic activity and complement activation. While highly immunogenic, pneumolysin induces erythrocyte lysis, which may limit its use in vaccines. Therefore, in some embodiments, the Ply polypeptide antigen is or includes a substantially non-hemolytic Ply variant (also referred herein as variant Ply or "mPly"). As used herein, the term "substantially non-hemolytic" means that its ability to lyse erythrocytes is 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% compared to the ability of a comparable concentration of reference Ply (e.g., wild-type Ply). In some embodiments, the 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%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% lower than that of a reference Ply (e.g., wild-type Ply) at an equivalent concentration. In some embodiments, substantially non-hemolytic Ply has no detectable hemolytic activity. The term “wild-type Ply” is defined according to the usual definitions associated with such terms, and in some embodiments, for example, is naturally occurring Ply (e.g., Ply naturally secreted by a viable bacterial source). In some embodiments, the wild-type Ply protein is represented by the amino acid sequence described in SEQ ID NO: 4.

[0114] In some embodiments, mutant Ply (e.g., non-hemolytic Ply) includes a wild-type Ply amino acid sequence (e.g., the amino acid sequence described in SEQ ID NO: 4) or an antigenic fragment thereof with one or more amino acid substitutions. In some embodiments, mutant Ply (e.g., non-hemolytic Ply) includes a wild-type Ply amino acid sequence (e.g., the amino acid sequence described in SEQ ID NO: 4) or an antigenic fragment thereof with one or more of the following amino acid substitutions: D385 residue substituted with N, C428 residue substituted with G, and W433 residue substituted with F (see, for example, Berry et al., "Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus neumoniae." Infect Immune 1995 63(5):1969-1974). In some embodiments, mutant Ply having amino acid substitutions D385N, C428G, and W433F (e.g., non-hemolytic Ply) is referred to as PdT. In some embodiments, PdT is or contains the amino acid sequence described in Sequence ID No. 5.

[0115] In some embodiments, mutant Ply (e.g., non-hemolytic Ply) includes the wild-type Ply amino acid sequence (e.g., the amino acid sequence described in SEQ ID NO: 4) or an antigenic fragment thereof, with the substitution of G294 to P (see, for example, Oloo et al., "Structure-guided antigen engineering yields pneumolysin mutants suitable for vaccination against pneumococcal disease". J Biol Chem. 2011 286(14):12133~12140). In some embodiments, mutant Ply (e.g., non-hemolytic Ply) includes the wild-type Ply amino acid sequence (e.g., the amino acid sequence described in SEQ ID NO: 4) or an antigenic fragment thereof, with all of the following amino acid substitutions: D385 is substituted with N, C428 is substituted with G, W433 is substituted with F, and G294 is substituted with P. In some embodiments, mutant Ply (e.g., non-hemolytic Ply) having amino acid substitutions G294P, D385N, C428G, and W433F is referred to as PdT(G294P). In some embodiments, PdT(G294P) is or contains the amino acid sequence described in Sequence ID No. 6.

[0116] In some embodiments, the mutant Ply (e.g., non-hemolytic Ply) is a portion of the PdT(G294P) polypeptide (e.g., a portion of the PdT(G294P) polypeptide of SEQ ID NO: 6, which comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or more consecutive amino acids of SEQ ID NO: 6). In some embodiments, such a portion of the PdT(G294P) polypeptide comprises the four amino acid substitutions described herein. In some embodiments, the portion of the PdT(G294P) polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence described in SEQ ID NO: 6. In some embodiments, mutant Ply (e.g., non-hemolytic Ply) contains one or more amino acid changes (e.g., deletions, substitutions, and / or insertions) from the PdT(G294P) polypeptide sequence of SEQ ID NO: 6. For example, mutant Ply (e.g., non-hemolytic Ply) may contain at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of an amino acid sequence 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 represented in SEQ ID NO: 6). Alternatively, mutant Ply (e.g., non-hemolytic Ply) may contain a portion of the sequence that is identical to SEQ ID NO: 6 by at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or more consecutive amino acids).In some embodiments, mutant Ply (e.g., non-hemolytic Ply) may contain 25 or fewer amino acid modifications (e.g., deletions, substitutions, and / or insertions) within the sequence of SEQ ID NO: 6 or a portion thereof as described herein (e.g., including 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, and 2 or fewer). In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions.

[0117] Exemplary pneumococcal polypeptide antigen: SP0435 polypeptide In some embodiments, the antigenic polypeptide described herein is or includes the pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or includes the SP0435 polypeptide. SP0435 is a protein of Streptococcus pneumoniae. In some embodiments, the SP0435 polypeptide is elongation factor P. In some embodiments, the SP0435 polypeptide is or includes the full-length SP0435 polypeptide. For example, in some embodiments, the full-length SP0435 polypeptide has 186 amino acids (20 kDa) and is represented by the amino acid sequence described in SEQ ID NO: 8. While we do not wish to adhere to any particular theory, amino acids 1-61 of SEQ ID NO: 8 (amino acids 1-61 of the full-length protein) are predicted to be the dimerization domain of the SP0435 polypeptide. Therefore, in some embodiments, the SP0435 polypeptide does not need to contain such a dimerization domain, for example, in some embodiments, the possibility of crosslinking or interference with the rizavidin moiety is minimized or avoided. In some embodiments, the SP0435 polypeptide includes a portion of the SP0435 polypeptide (for example, the portion of the SP0435 polypeptide of SEQ ID NO: 8, which 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 consecutive amino acids of SEQ ID NO: 8). In some embodiments, the portion of the SP0435 polypeptide corresponds to a protein having amino acids 62-185 of the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the SP0435 polypeptide contains one or more amino acid changes (e.g., deletions, substitutions, and / or insertions) from the naturally occurring wild-type SP0435 polypeptide sequence.For example, SP0435 polypeptide may contain at least 60% (for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of an amino acid sequence identical to SEQ ID NO: 8 or a portion thereof (for example, 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 represented in SEQ ID NO: 8). Alternatively, the SP0435 polypeptide may contain at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of a sequence identical to SEQ ID NO: 8 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or more consecutive amino acids). In some embodiments, the nucleotide sequence encoding the SP0435 polypeptide is provided herein as SEQ ID NO: 17. In some embodiments, the SP0434 polypeptide may contain 25 or fewer amino acid modifications (e.g., deletions, substitutions, and / or insertions) within the sequence of SEQ ID NO: 8 or a portion thereof as described herein (e.g., including 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, and 2 or fewer). In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions. In some embodiments, the SP0435 polypeptide is described in WO 2011 / 112906 (the entire contents of which are incorporated herein by reference for the purposes described herein).

[0118] Exemplary pneumococcal polypeptide antigen: SP0785 polypeptide In some embodiments, the antigenic polypeptide described herein is or includes the pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or includes the SP0785 polypeptide. SP0785 is a conserved hypothetical Streptococcus pneumoniae protein, such as, for example, in some embodiments, described in WO2014 / 124228 (for the purposes described herein, the entire contents thereof are incorporated herein by reference). In some embodiments, the SP0785 polypeptide is an efflux transporter protein conserved among strains of Streptococcus pneumoniae. In some embodiments, the SP0785 polypeptide is or includes the full-length SP0785 polypeptide. For example, in some embodiments, the full-length SP0785 polypeptide has 399 amino acids (38 kDa) and is represented by the amino acid sequence described in SEQ ID NO: 10. While we do not wish to adhere to any particular theory, amino acids 1-32 of SEQ ID NO: 10 (amino acids 1-32 of the full-length protein) are predicted to be the signal sequence and transmembrane domain of the SP0785 polypeptide. Therefore, in some embodiments, the SP0785 polypeptide does not need to include such a signal sequence and transmembrane domain. In some embodiments, the SP0785 polypeptide includes a portion of the SP0785 polypeptide (for example, a portion of the SP0785 polypeptide of SEQ ID NO: 10, which 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 consecutive amino acids of SEQ ID NO: 10). In some embodiments, the SP0785 polypeptide portion corresponds to a protein having amino acids 33-399 of the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the SP0785 polypeptide contains one or more amino acid changes (e.g., deletions, substitutions, and / or insertions) from the naturally occurring wild-type SP0785 polypeptide sequence.For example, SP0785 polypeptide may contain at least 60% (for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of an amino acid sequence identical to SEQ ID NO: 10 or a portion thereof (for example, 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 represented in SEQ ID NO: 10). Alternatively, the SP0785 polypeptide may contain at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of a sequence identical to SEQ ID NO: 10 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids). In some embodiments, the nucleotide sequence encoding the SP0785 polypeptide is provided herein as SEQ ID NO: 18. In some embodiments, the SP0785 polypeptide may contain 25 or fewer amino acid modifications (e.g., deletions, substitutions, and / or insertions) within the sequence of SEQ ID NO: 10 or a portion thereof as described herein (e.g., 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, and 2 or fewer). In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions.

[0119] Exemplary pneumococcal polypeptide antigen; SP1500 polypeptide In some embodiments, the antigenic polypeptide described herein is or includes the pneumococcal polypeptide antigen. In some embodiments, the pneumococcal polypeptide antigen is or includes the SP1500 polypeptide. SP1500 is a protein of Streptococcus pneumoniae, such as, for example, in some embodiments, described in WO 2014 / 124228 (for the purposes described herein, the entire content of which is incorporated herein by reference). In some embodiments, the SP1500 polypeptide is an amino acid ABC transporter, an amino acid-linked polypeptide conserved among strains of Streptococcus pneumoniae. In some embodiments, the SP1500 polypeptide is or includes the full-length SP1500 polypeptide. For example, in some embodiments, the full-length SP1500 polypeptide has 278 amino acids (28 kDa) and is represented by the amino acid sequence described in SEQ ID NO: 12. While we do not wish to adhere to any particular theory, amino acids 1-26 of SEQ ID NO: 12 (amino acids 1-26 of the full-length protein) are predicted to be the signal sequence of the SP1500 polypeptide. Therefore, in some embodiments, the SP1500 polypeptide does not need to include such a signal sequence. In some embodiments, the SP1500 polypeptide includes a portion of the SP1500 polypeptide (for example, the portion of the SP1500 polypeptide of SEQ ID NO: 12, which 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 consecutive amino acids of SEQ ID NO: 12). In some embodiments, the portion of the SP1500 polypeptide corresponds to a protein having amino acids 27-278 of the amino acid sequence described in SEQ ID NO: 12. In some embodiments, the SP1500 polypeptide contains one or more amino acid changes (e.g., deletions, substitutions, and / or insertions) from the naturally occurring wild-type SP1500 polypeptide sequence.For example, the SP1500 polypeptide may contain at least 60% (for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) of an amino acid sequence identical to SEQ ID NO: 12 or a portion thereof (for example, 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 represented in SEQ ID NO: 12). Alternatively, the SP1500 polypeptide may contain at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) of a sequence identical to SEQ ID NO: 12 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or more consecutive amino acids). In some embodiments, the nucleotide sequence encoding the SP1500 polypeptide is provided herein as SEQ ID NO: 19. In some embodiments, the SP1500 polypeptide may contain 25 or fewer amino acid modifications (e.g., deletions, substitutions, and / or insertions) within the sequence of SEQ ID NO: 12 or a portion thereof as described herein (e.g., including 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, and 2 or fewer). In some embodiments, such amino acid modifications may be present in the N-terminal and / or C-terminal portions.

[0120] In some embodiments, the nucleic acid sequences encoding the pneumolysin polypeptide (SEQ ID NO: 4), SP0435 polypeptide (SEQ ID NO: 8), SP0785 polypeptide (SEQ ID NO: 10), and SP1500 polypeptide (SEQ ID NO: 12) are provided as SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. Those skilled in the art will understand, by genetic coding degeneracy, that other DNA sequences (including codon-optimized sequences) may encode these polypeptides and others disclosed herein.

[0121] In some embodiments, this disclosure includes nucleic acid sequences encoding any of the amino acids described herein. Those skilled in the art will understand, by the degeneracy of the genetic code, that other DNA sequences (including codon-optimized sequences) may encode these polypeptides and other materials disclosed herein.

[0122] Fusion protein containing antigenic polypeptide The antigenic polypeptides described herein may be parts of a fusion protein. For example, in some embodiments, the immunogenic complex described herein comprises a complementary affinity molecule described herein and a fusion protein which is or contains one or more antigenic polypeptides. In some embodiments, the fusion protein comprises two antigenic polypeptides described herein. In some embodiments, the fusion protein comprises three antigenic polypeptides described herein. In some embodiments, the fusion protein comprises four antigenic polypeptides described herein. In some embodiments, the fusion protein comprises 6 to 10 antigenic polypeptides described herein. In some embodiments, the fusion protein comprises 11 to 15 antigenic polypeptides described herein. In some embodiments, the fusion protein comprises 15 to 20 antigenic polypeptides described herein.

[0123] In some embodiments, the fusion protein has carrier properties. In some embodiments, the fusion protein has antigenic properties. In some embodiments, the fusion protein has both carrier and antigenic properties.

[0124] In some embodiments, the fusion protein comprises at least one antigenic polypeptide of a bacterium, fungus, parasite, virus, or cancer or tumor, or an antigenic polypeptide derived therefrom. In some embodiments, the fusion protein comprises a Streptococcus pneumoniae polypeptide antigen or an antigenic polypeptide containing the same (for example, in some embodiments, pneumolysin polypeptide antigen, SP0435 polypeptide antigen, SP0785 polypeptide antigen, or SP1500 polypeptide antigen).

[0125] In some embodiments, the fusion protein contains one or more antigenic polypeptides or antigenic fragments thereof of Streptococcus pneumoniae having amino acid sequences identical by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% to any of SEQ ID NOs: 4 to 13. In some embodiments, the fusion protein contains one or more antigenic polypeptides or antigenic fragments thereof of Streptococcus pneumoniae having amino acid sequences including any of SEQ ID NOs: 4 to 13. In some embodiments, the fusion protein contains two antigenic polypeptides or antigenic fragments thereof having amino acid sequences identical by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% to any of SEQ ID NOs: 4 to 13. In some embodiments, the fusion protein contains two antigenic polypeptides or antigenic fragments thereof having amino acid sequences including any of SEQ ID NOs: 4 to 13. In some embodiments, the fusion protein described herein further comprises a biotin-binding site, for example, in some embodiments, a rizavidin polypeptide having an amino acid sequence identical to 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% of any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof.

[0126] In some embodiments, the fusion protein comprises a pneumolysin polypeptide as described herein. For example, in some embodiments, the fusion protein comprises a polypeptide or antigenic fragment thereof having 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 the Streptococcus pneumoniae PdT(G294P) polypeptide). In some embodiments, the pneumolysin polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rizavidin polypeptide, for example, in some embodiments, a rizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3 or a biotin-bound fragment thereof.

[0127] In some embodiments, the fusion protein comprises the SP0435 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide or antigenic fragment thereof having 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 Streptococcus pneumoniae SP0435 polypeptide). In some embodiments, the SP0435 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rizavidin polypeptide, for example, in some embodiments, a rizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3 or a biotin-bound fragment thereof.

[0128] In some embodiments, the fusion protein comprises the SP0785 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide or antigenic fragment thereof having 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 the Streptococcus pneumoniae SP0785 polypeptide). In some embodiments, the SP0785 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rizavidin polypeptide, for example, in some embodiments, a rizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3 or a biotin-bound fragment thereof.

[0129] In some embodiments, the fusion protein comprises the SP1500 polypeptide. For example, in some embodiments, the fusion protein comprises a polypeptide or antigenic fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 13 (amino acids 27-278 of the Streptococcus pneumoniae SP1500 polypeptide). In some embodiments, the SP1500 polypeptide described herein may be truncated from its N-terminal and / or C-terminal portions. In some embodiments, the fusion protein as described herein further comprises a rizavidin polypeptide, for example, in some embodiments, a rizavidin polypeptide having the amino acid sequence of any of SEQ ID NOs: 1-3 or a biotin-bound fragment thereof.

[0130] In some embodiments, the fusion protein comprises one or more polypeptides homologous to the polypeptides described herein (e.g., antigenic polypeptides isolated from various serotypes, strains, or species). Individual serotypes, strains, or species may contain numerous mutations relative to one another, some of which may result in different protein sequences among the various serotypes, strains, or species. In some embodiments, the fusion protein comprises one or more polypeptides homologous to the Streptococcus pneumoniae polypeptides described herein, e.g., Ply polypeptide, SP0435 polypeptide, SP0785 polypeptide, or SP1500 polypeptide isolated from various serotypes of Streptococcus pneumoniae. In some embodiments, the antigenic polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any polypeptide or antigenic fragment thereof of SEQ ID NOs: 4-13. Differences in serotype, strain, or species may be used to design such variants of the antigenic polypeptides described herein.

[0131] In some embodiments, the fusion protein described herein comprises one or more fragments of one or more polypeptides described herein, for example, a biotin-bound fragment of rizavidin or an antigenic fragment of an antigenic polypeptide (e.g., Ply polypeptide or its antigenic fragment, SP0435 polypeptide or its antigenic fragment, SP0785 polypeptide or its antigenic fragment (e.g., with or without a signal sequence), or SP1500 polypeptide or its antigenic fragment (e.g., with or without a signal sequence)). In some embodiments, the fusion protein described herein comprises a truncated form of a polypeptide described herein, a truncated form that is close in size to a polypeptide having any of the amino acid sequences of SEQ ID NOs. 4 to 13. For example, in some embodiments, such a truncated polypeptide may have up to 1, 2, 3, 4, 5, 10, or 20 amino acids missing from one or both ends of the original polypeptide. In some embodiments, the same number of residues are removed from the N-terminus and C-terminus, while in other embodiments, a different number of residues are removed from the N-terminus compared to the C-terminus. In some embodiments, the truncated polypeptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any of SEQ ID NOs: 4-13, which lacks 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both.10 amino acid residues from the N-terminus, C-terminus, or both.In some embodiments, the truncated polypeptide has one of the amino acid sequences of SEQ ID NOs: 4-13, which is missing 1-10 amino acid residues from the N-terminus, C-terminus, or both. For example, the truncated polypeptide may be a protein missing 20 amino acid residues by deleting 10 amino acid residues from both the N-terminus and C-terminus of one of the SEQ ID NOs: 4-13. For example, internal deletions of amino acids 1-10, 11-20, 21-30, or 31-40 are also intended.

[0132] In some embodiments, the fusion protein comprises an N-terminal polypeptide and a C-terminal polypeptide. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide are antigenic polypeptides described herein (e.g., polypeptides having an amino acid sequence including one or more of SEQ ID NOs: 4-13) or antigenic fragments or variants thereof. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide are polypeptides having an amino acid sequence including a biotin-binding site, e.g., SEQ ID NOs: 1, SEQ ID NOs: 2, or SEQ ID NOs: 3, or biotin-binding fragments thereof. In some embodiments, one of the N-terminal polypeptide or the C-terminal polypeptide is a polypeptide having an amino acid sequence including a biotin-binding site, e.g., SEQ ID NOs: 1, SEQ ID NOs: 2, or SEQ ID NOs: 3, or biotin-binding fragments thereof, and the other-terminal polypeptide is an antigenic polypeptide (e.g., polypeptides having an amino acid sequence including one or more of SEQ ID NOs: 4-13) or antigenic fragments or variants thereof.

[0133] In some embodiments, the N-terminal and C-terminal polypeptides present in the fusion protein are directly linked to each other. In some embodiments, the N-terminal and C-terminal polypeptides present in the fusion protein are linked via a linker peptide (e.g., a linker disclosed herein). The length and / or amino acids of the linker, if present, can be adjusted to obtain a more flexible, semi-rigid, or rigid linker. In some embodiments, the linker may be GS-rich. In some embodiments, the linker may be A-rich. The linker may generally have an amino acid length of 1 to 40, for example, 3 to 10 or 10 to 30, and specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Exemplary flexible peptide linkers are represented as SEQ ID NOs. 38-42 and 45-47.

[0134] In some embodiments, the fusion protein includes one or more linkers as described herein. In some embodiments, the fusion protein includes at least one linker. In some embodiments, the fusion protein includes at least two linkers. In some embodiments, the linker includes a polypeptide comprising the amino acid sequence GGGGSSS (SEQ ID NO: 38). In some embodiments, the linker includes a polypeptide comprising the amino acid sequence AAA. In some embodiments, the linker includes a polypeptide comprising the amino acid sequence AAAA (SEQ ID NO: 66). In some embodiments, the linker includes a polypeptide comprising the amino acid sequence GGGSGGGGSGGGGS (SEQ ID NO: 41). In some embodiments, the fusion protein includes a first linker having the amino acid sequence GGGGSSS (SEQ ID NO: 38) and a second linker having the amino acid sequence AAA. In some embodiments, the linker sequence may be a residual amino acid sequence from a restriction enzyme site. For example, in some embodiments, the fusion protein includes the residual amino acid sequence AAA from a NotI restriction enzyme site. In some embodiments, the fusion protein includes the GGGGSSS (SEQ ID NO: 38) linker and the residual amino acid sequence AAA from a NotI restriction enzyme site. In some embodiments, one or more linkers are selected from GGGGSSS (SEQ ID NO: 38) and AAAA (SEQ ID NO: 66). In some embodiments, the fusion protein includes the linkers GGGGSSS (SEQ ID NO: 38) and AAAA (SEQ ID NO: 66).

[0135] In some embodiments, the fusion protein described herein includes a biotin-binding site. In some embodiments, the fusion protein includes a biotin-binding site and one or more polypeptide antigens. In some embodiments, the fusion protein includes a biotin-binding site and two or more polypeptide antigens. In some embodiments, the biotin-binding site of the fusion protein includes rizavidin or a biotin-binding fragment thereof, which in some embodiments is further described in WO 2012 / 155053 (for the purposes described herein, this content is incorporated herein by reference in its entirety).

[0136] In some embodiments, the fusion protein described herein includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof. In some embodiments, the fusion protein includes a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 2 (denoted as Rhavi, amino acids 45-179 of rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof.

[0137] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 7 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) SEQ ID NO: 7 (iii) A polypeptide or antigenic fragment thereof 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 with the sequence of (amino acids 2-470 of the Streptococcus pneumoniae PdT(G294P) polypeptide), and (iii) a polypeptide or antigenic fragment thereof 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 with the sequence of SEQ ID NO: 9 (amino acids 62-185 of the Streptococcus pneumoniae SP0435 polypeptide). 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 one amino acid sequence from SEQ ID NOs: 38-67 and AAA. In some embodiments, each of these linkers is GGGGSSS (Sequence ID 38).

[0138] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or less identity with the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785 polypeptide). (iii) a polypeptide or antigenic fragment thereof containing an amino acid sequence having at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 13 (amino acids 27-278 of the Streptococcus pneumoniae SP1500 polypeptide), and (iii) a polypeptide or antigenic fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 13 (amino acids 27-278 of the Streptococcus pneumoniae SP1500 polypeptide). 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 one amino acid sequence from 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 contains the amino acid sequence AAA remaining from the NotI restriction enzyme site. In some embodiments, the fusion protein includes a linker of amino acid sequence AAA remaining from GGGGSSS (SEQ ID NO: 38) and the NotI restriction enzyme site.

[0139] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (denoted as Rhavi, amino acids 45-179 of rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, or less identity with the sequence of SEQ ID NO: 7 (amino acids 2-470 of the Streptococcus pneumoniae PdT(G294P) polypeptide) (iii) A polypeptide or antigenic fragment thereof comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, and (iii) a polypeptide or antigenic fragment thereof 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 the Streptococcus pneumoniae SP0435 polypeptide). 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 one amino acid sequence from 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, the fusion protein described herein includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein includes the amino acid sequence of 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.

[0140] In some embodiments, the fusion proteins described herein include: (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 2 (denoted as Rhavi, amino acids 45-179 of rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 11 (amino acids 33-399 of the Streptococcus pneumoniae SP0785 polypeptide) (iii) A polypeptide or antigenic fragment thereof comprising an amino acid sequence having 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, and (iii) a polypeptide or antigenic fragment thereof 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 the Streptococcus pneumoniae SP1500 polypeptide). In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, one or more such linkers are independently selected from the group consisting of one amino acid sequence from SEQ ID NOs: 38-67 and AAA. In some embodiments, one or more such linkers are selected from GGGGSSS (SEQ ID NO: 38) and AAA. In some embodiments, the fusion protein includes the amino acid sequence AAA remaining from the NotI restriction enzyme site. In some embodiments, the fusion protein includes a linker of GGGGSSS (SEQ ID NO: 38) and the amino acid sequence AAA remaining from the NotI restriction enzyme site.In some embodiments, the fusion protein described herein includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 30 (CP1) or SEQ ID NO: 31.

[0141] In some embodiments, the Disclosure provides a fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to a fusion protein having any one of the amino acid sequences of SEQ ID NOs. 20 to 31. In some embodiments, the fusion protein is 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 NOs. 21, or includes such an amino acid sequence. In some embodiments, the fusion protein is 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, the fusion protein is or contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein is or contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to CP1.

[0142] In some embodiments, the fusion protein described herein comprises an antigenic fragment of a fusion protein having one of the amino acid sequences of SEQ ID NOs. 20-31. For example, the fusion protein may lack up to 1, 2, 3, 4, 5, 10, or 20 amino acids from one of the N-terminus, C-terminus, or both of SEQ ID NOs. 20-31. In some embodiments, the same number of residues are removed from the N-terminus and C-terminus, while in other embodiments, a different number of residues are removed from the N-terminus compared to the C-terminus. In some embodiments, the fusion protein is or contains the antigenic fragment of SEQ ID NOs. 20 or SEQ ID NOs. 21. In some embodiments, the fusion protein is or contains the antigenic fragment of SPP2. In some embodiments, the fusion protein is or contains the antigenic fragment of SEQ ID NOs. 30 or SEQ ID NOs. 31. In some embodiments, the fusion protein is or contains the antigenic fragment of CP1.

[0143] In some embodiments, the fusion proteins described herein include a variant or fragment of a polypeptide having the amino acid sequence of SEQ ID NOs: 1 to 13. In some embodiments, the fusion proteins described herein include a polypeptide encoded by a variant or fragment of a gene having the nucleic acid sequence of SEQ ID NOs: 1 to 13. In some embodiments, the fragments contained in the fusion proteins described herein are close in size to the full-length polypeptide or the polypeptide having the amino acid sequence of SEQ ID NOs: 1 to 13. For example, the fragment may be missing up to 1, 2, 3, 4, 5, 10, 20, or 30 amino acids from one or both ends. In some embodiments, the fragment is 25 to 50 amino acid long, or 50 to 100, or 100 to 150, or 150 to 200, or 200 to 250, or 250 to 300, or 300 to 350 amino acid long. In some embodiments, the fragments are obtained from treatment or partial treatment of a signal sequence by an expression host, e.g., Escherichia coli, an insect cell line (e.g., a baculovirus expression system), a yeast cell line (e.g., S. cerevisiae or S. pombe), or a mammalian cell line (e.g., human or Chinese hamster ovary). The above fragments or sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the following biological activities, e.g., a biological activity that increases the amount of IL-17 released by at least 1.5 times or 2 times, or more (e.g., in absolute terms or relative to a control protein).

[0144] In some embodiments, the fusion proteins described herein may be useful in the immunogenic complexes described herein.

[0145] Linker or spacer In some embodiments, the polypeptides described herein are linked to one or more target entities (e.g., small molecules, polypeptides, polynucleotides, lipids, polysaccharides, etc.) by one or more linkers. In some embodiments, the fusion proteins described herein include one or more linkers. In some embodiments, the linkers are one or more amino acids or include one or more amino acids. In some embodiments, the fusion protein includes an antigenic polypeptide linked to a biotin-binding site by a linker. In some embodiments, the fusion protein includes a first antigenic polypeptide, a second antigenic polypeptide, a biotin-binding site, and at least one linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are linked by a linker. In some embodiments, the first antigenic polypeptide or the second antigenic polypeptide is linked to a biotin-binding site by a linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are linked by a first linker, and the first antigenic polypeptide or the second antigenic polypeptide is linked to a biotin-binding site by a second linker.

[0146] In some embodiments, the fusion protein described herein comprises a first linker containing the amino acid sequence GGGGSSS (SEQ ID NO: 38) and a second linker containing the amino acid sequence GGGGSSS (SEQ ID NO: 38). In some embodiments, the fusion protein described herein comprises a first linker containing the amino acid sequence GGGGSSS (SEQ ID NO: 38) and a second linker containing the amino acid sequence AAA.

[0147] In some embodiments, the linker interposes a structure between two protein sites. In some embodiments, the structure is or includes an α-helix. In some embodiments, the structure is or includes a β-strand. In some embodiments, the structure is or includes a coil / bend. In some embodiments, the structure is or includes a turn. In some embodiments, the linker reduces steric hindrance between the two protein sites linked by the linker. In some embodiments, the linker reduces undesirable interactions between the two protein sites linked by the linker. In some embodiments, the linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may additionally include threonine, proline, and / or alanine residues. In some embodiments, the linker is hydrophilic. In some embodiments, the linker is hydrophobic. In some embodiments, the linker increases the stability of the fusion protein containing the linker.

[0148] In some embodiments, the linker does not interfere with the folding or activity of the polypeptide to which it is intended to be linked (e.g., an antigenic polypeptide in some embodiments). In some embodiments, the linker does not interfere with the antigenicity of the antigenic polypeptide to which it is linked. In some embodiments, the linker does not reduce the antigenicity of the antigenic polypeptide to which it is linked. In some embodiments, the linker does not cause the antigenicity of the antigenic polypeptide to which it is linked to to be lost. In some embodiments, the effect of the linker is determined by comparing the polypeptide with the polypeptide to which the linker is linked.

[0149] In some embodiments, the linker does not interfere with the folding of the biotin-binding site to which it is linked. In some embodiments, the linker does not interfere with the biotin-binding capacity of the biotin-binding site to which it is linked. In some embodiments, the linker does not reduce the biotin-binding capacity of the biotin-binding site to which it is linked. In some embodiments, the linker does not eliminate the biotin-binding capacity of the biotin-binding site to which it is linked. In some embodiments, the effect of the linker is determined by comparing the biotin-binding site with the biotin-binding site to which the linker is linked.

[0150] In some embodiments, the linker is not antigenic. In some embodiments, the linker does not induce a T cell response. In some embodiments, the linker does not induce a B cell response. In some embodiments, the linker does not induce a T cell or B cell response.

[0151] In some embodiments, the linker contains two or more amino acids. In some embodiments, the linker may be 3-100, 5-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, or 2-3 amino acid lengths. In some embodiments, the linker contains amino acids between 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, or 10-15. In some embodiments, the linker contains at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, the linker is or includes a peptidyl linker containing 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.

[0152] In some embodiments, the linker is a mobile linker. A mobile linker may be useful for linking domains that require some degree of movement or interaction, and may contain small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Ser or Thr incorporation can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing undesirable interactions between the linker and protein sites. In some embodiments, the linker contains small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. In some embodiments, the linker is a Gly-Ser linker.

[0153] In some embodiments, the linker is or contains the amino acid sequence GGGGSSS (SEQ ID NO: 38). In some embodiments, the linker is (GGGGS) n The sequence is or includes (SEQ ID NO: 39), where n represents the number of repeats in the GGGGS (SEQ ID NO: 40) unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the polypeptide linker may have an amino acid sequence that is or includes GGGGSGGGGSGGGGS (SEQ ID NO: 41) (i.e., (GGGGS)3) or GGGGSGGGGSGGGGSGGGGSGGGGGSGGGGGS (SEQ ID NO: 42) (i.e., (GGGGS)6). In some embodiments, the linker includes one or more of Gly, Ser, Thr, Ala, Lys, and Glu. In some embodiments, the linker is or includes KESGSVSSEQLAQFRSLD (SEQ ID NO: 43). In some embodiments, the linker is or includes EGKSSGSGSESKST (SEQ ID NO: 44). In some embodiments, the linker is (Gly) nThe linker is (SEQ ID NO: 45) or includes it, where n is the number of repeating Gly residues, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the linker is GGG or includes it. In some embodiments, the linker is (Gly)6 (SEQ ID NO: 46) or includes it. In some embodiments, the linker is (Gly)8 (SEQ ID NO: 47) or includes it. In some embodiments, the linker is GSAGSAAGSGEF (SEQ ID NO: 48) or includes it. In some embodiments, the linker is amino acid sequence AAA or includes it. In some embodiments, such a linker may be synthesized or derived from amino acid residues from restriction enzyme sites (e.g., Not I restriction enzyme sites).

[0154] In some embodiments, the linker is a rigid linker. A rigid linker is useful for fixing and maintaining the distance between domains and preserving their independent functions. A rigid linker may also be useful when the spatial separation of domains is important for preserving the stability or bioactivity of one or more components of the fusion. In some embodiments, the linker is (EAAAK) n (Sequence ID 49) or including the same, where n represents the number of repetitions in the EAAAK (Sequence ID 50) unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is A(EAAAK) n A (Sequence ID 51) or including A, where n represents the number of repetitions in the EAAAK (Sequence ID 50) unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is A(EAAAK) nA (SEQ ID NO: 52) or includes this, where n represents the number of repetitions of the EAAAK (SEQ ID NO: 50) unit, and n is 2, 3, 4, or 5. In some embodiments, the linker is A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 53) or includes this. In some embodiments, the linker is [A(EAAAK) n A] m (SEQ ID NO: 54) or includes this, where n is 2, 3, or 4, and m is 1 or 2. In some embodiments, the linker is AEAAAKEAAAKA (SEQ ID NO: 55) or includes this.

[0155] In some embodiments, the linker is (X-Pro) n (SEQ ID NO: 56) or includes this, X designates any amino acid, where n represents the number of repetitions of the X-Pro unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the linker is (Ala-Pro) n (SEQ ID NO: 57) or includes this, where n represents the number of repetitions of the Ala-Pro unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the linker is (Ala-Pro) n (SEQ ID NO: 58) or includes this, where n represents the number of repetitions of the Ala-Pro unit, and n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.

[0156] In some embodiments, the linker is (Lys-Pro) n (SEQ ID NO: 59) or includes this, where n represents the number of repetitions of the Lys-Pro unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, or more. In some embodiments, the linker is (Glu-Pro)n The linker is (SEQ ID NO: 60) or includes it, where n represents the number of repeating units of Glu-Pro, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the linker is (Ala-Pro)7 (SEQ ID NO: 61) or includes it.

[0157] In some embodiments, the linker is or includes GAPGGGGGAAAAAGGGGGGAP (GAG linker, SEQ ID NO: 62). In some embodiments, the linker is or includes GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 linker, SEQ ID NO: 63). In some embodiments, the linker is or includes GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG3 linker, SEQ ID NO: 64).

[0158] In some embodiments, the linker is or includes a VSDP (SEQ ID NO: 65).

[0159] In some embodiments, the linker is AAAA (SEQ ID NO: 66) or includes it.

[0160] In some embodiments, the linker is GGGG (SEQ ID NO: 67) or includes it.

[0161] Suitable linkers or spacers also include those having amino acid sequences that are 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 exemplary linkers described above. In some embodiments, the linker includes a polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of GGGGSSS (SEQ ID NO: 38).

[0162] Additional linkers suitable for use in certain embodiments are found in U.S. Patent Publication No. 2012 / 0232021, filed March 2, 2012, and [Chen, 2013], the disclosures thereof which are incorporated herein by reference in their entirety.

[0163] tag In some embodiments, the polypeptides described herein (e.g., fusion proteins) may comprise one or more tags. The tags may be located at the N-terminus or C-terminus of the polypeptide. For example, the tags may be attached to the polypeptide (e.g., via addition or modification to the encoding DNA sequence) to facilitate purification, detection, solubility, or to confer other desired properties to the polypeptide. In some embodiments, the tags may be peptides, oligopeptides, or polypeptides that can be used for affinity purification. In some embodiments, the tag is one or more of polyhistidine (His), glutathione S-transferase (GST), tandem affinity purified protein (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose-binding protein (MBP), vesicular stomatitis virus glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), calmodulin, Nus, S-tag, lipoprotein D, and galactosidase, or is derived from these. In some embodiments, the His tag is H n The amino acid sequence is or includes the sequence, where n is an integer from 2 to 10 (SEQ ID NO: 68). Exemplary His tags include HHHHHH (SEQ ID NO: 69) and MSYYHHHHHH (SEQ ID NO: 70). In other embodiments, the polypeptides described herein are purified by a method that does not include tags such as protein purification tags and does not depend on affinity to purification tags. In some embodiments, the fusion proteins described herein include (i) a polypeptide of any amino acid sequence of SEQ ID NOs: 4 to 13, or a fusion protein of any amino acid sequence of SEQ ID NOs: 20 to 31, and (ii) a tag having 1, 2, 3, 4, 5, 10, or 20 or fewer amino acids at one or both ends of the polypeptide or fusion protein of (i).

[0164] In some embodiments, the polypeptides described herein may contain membrane translocation sequences (MTS) that, for example, facilitate the introduction of fusion proteins into mammalian cells and subsequent stimulation of cell-mediated immune responses. Exemplary membrane translocation sequences include, but are not limited to, the hydrophobic region of the signal sequence of Kaposi's fibroblast growth factor, the MTS of synuclein, the third helix of the Antennapedia homeodomain, SN50, the integrin 3h region, Tat, pAntp, PR-39 of HIV, abaecin, apidesin, Bac5, Bac7, the CS protein of P. berghei, and the MTS described in U.S. Patent Publications 6,248,558; 6,432,680; and 6,248,558.

[0165] In some embodiments, the polypeptides described herein (e.g., fusion proteins) may include one or more tags. The tags may be N-terminus or C-terminus. For example, the tags may be attached to a polypeptide (e.g., via addition or modification to an encoding DNA sequence) to facilitate purification, detection, solubility, or to confer other desired properties to the protein. In some embodiments, the tags may be peptides, oligopeptides, or polypeptides that can be used for affinity purification. In some embodiments, the tag is one or more of polyhistidine (His), glutathione S-transferase (GST), tandem affinity purified protein (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose-binding protein (MBP), vesicular stomatitis virus glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), calmodulin, Nus, S-tag, lipoprotein D, and galactosidase, or is derived from these. In some embodiments, the His tag is H nThe amino acid sequence is or includes the sequence, where n is an integer from 2 to 10 (SEQ ID NO: 68). Exemplary His tags include HHHHHH (SEQ ID NO: 69) and MSYYHHHHHH (SEQ ID NO: 70). In other embodiments, the fusion proteins described herein are purified by a method that does not include tags such as protein purification tags and is independent of affinity to purification tags. In some embodiments, the fusion proteins described herein include (i) a polypeptide of any amino acid sequence of SEQ ID NOs: 1 to 13 or a fusion protein of any amino acid sequence of SEQ ID NOs: 20 to 31, and (ii) a tag having 1, 2, 3, 4, 5, 10, or 20 or fewer amino acids at one or both ends of the polypeptide or fusion protein of (i).

[0166] In some embodiments, the polypeptides described herein may contain membrane translocation sequences (MTS) that, for example, facilitate the introduction of fusion proteins into mammalian cells and subsequent stimulation of cell-mediated immune responses. Exemplary membrane translocation sequences include, but are not limited to, the hydrophobic region of the signal sequence of Kaposi's fibroblast growth factor, the MTS of synuclein, the third helix of the Antennapedia homeodomain, SN50, the integrin 3h region, Tat, pAntp, PR-39, abaesin, apidesin, Bac5, Bac7, the CS protein of P. bergey, and the MTS described in U.S. Patent Publications 6,248,558; 6,432,680; and 6,248,558.

[0167] Signal peptide In some embodiments, the polypeptides described herein include a signal peptide. In some embodiments, the fusion protein includes a signal peptide. The signal peptide, which may also be called a leader peptide, functions in the translocation of the polypeptide across the cell membrane (e.g., the cell membrane, endoplasmic reticulum, etc.). In some embodiments, the polypeptide includes the signal peptide at or near its N-terminus. In some embodiments, the signal sequence may or may not be cleaved after translocation across the membrane (e.g., by a signal peptidase).

[0168] The signal sequence may be linked directly to the polypeptide (e.g., by a peptide bond) or indirectly (e.g., by a linker). In some embodiments, the signal sequence may be linked to the polypeptide by a peptide linker disclosed herein.

[0169] In some embodiments, the signal peptide may be a bacterial signal peptide, a viral signal peptide, or a eukaryotic signal peptide. In some embodiments, the eukaryotic signal peptide is a mammalian signal peptide (e.g., a signal peptide from or derived from a human protein).

[0170] In some embodiments, the signal peptide may be a bacterial signal peptide. Bacterial signal peptides can be used to facilitate the translocation of polypeptides into the bacterial periplasmic space. In some embodiments, the signal peptide is cleaved from the polypeptide after translocation into the bacterial (e.g., E. coli) periplasmic space. Signal peptides directed at protein secretion from bacterial cells are known in the art and are disclosed, for example, in international application WO 2005 / 071088.

[0171] Any signal peptide known to those skilled in the art can be used. In some embodiments, the signal peptide is 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 The signal peptide is or includes the amino acid sequence of (SEQ ID NO: 80), MTDKKSENQTEKTETKENKGMTRREMLKLSAVAGTGIAVGATGLGTILNVVDQVDKALT (SEQ ID NO: 81), MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLGLGLTIAQSVGAFG (SEQ ID NO: 82), or a derivative thereof or functional moiety thereof. In some embodiments, the signal peptide is or includes the amino acid sequence of the human Ig heavy chain V-III region VH26 signal peptide, or a derivative thereof or functional moiety thereof. In some embodiments, the signal peptide is or includes the amino acid sequence of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 83), or a derivative thereof or functional moiety thereof. In some embodiments, the signal peptide is or includes the human IgG2 heavy chain signal peptide, or a derivative thereof or functional moiety thereof. In some embodiments, the signal peptide is or includes the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 84), or a derivative thereof or functional moiety thereof.

[0172] An example of a signal peptide can be found on the signal peptide database website, www.signalpeptide.de.

[0173] In some embodiments, the polypeptides described herein lack a signal peptide.

[0174] affinity molecular pairs As described herein, the immunogenic complexes of this disclosure include a complementary affinity molecule pair comprising an affinity molecule (i.e., a first affinity molecule) and a complementary affinity molecule (i.e., a second affinity molecule complementary to the first affinity molecule). In some embodiments, the affinity molecule / complementary affinity molecule pair is selected from one or more of the following: biotin / biotin binding sites, antibodies / antigens, enzymes / substrates, receptors / ligands, metals / metal-binding proteins, carbohydrates / carbohydrate-binding proteins, lipids / lipid-binding proteins, and His-tag / His-tag binding molecules. In some embodiments, the first affinity molecule interacts with a polymer (e.g., a polysaccharide, but not limited). In some embodiments, the second affinity molecule interacts with a polypeptide of interest (e.g., a polypeptide antigen, but not limited).

[0175] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is a biotin-binding site, for example, a biotin-binding protein or polypeptide, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the fusion protein of the immunogenic complex comprises a biotin-binding site and one or more polypeptide antigens. In some embodiments, the fusion protein comprises a biotin-binding site and two or more polypeptide antigens. In some embodiments, the biotin-binding site is a biotin-binding site capable of forming a dimer. In some embodiments, the biotin-binding site is a biotin-binding site capable of forming a tetramer. In some embodiments, the biotin-binding site is or comprises rizavidin, avidin, streptavidin, bladavidin, tamavidin, lentiavidin, zebavidin, neutraavidin, captoavidin (CaptAvidin®), or their biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is rizavidin, its biotin-binding domain, or a biotin-binding fragment, or includes thereof.

[0176] In some embodiments, the MAPS complex disclosed herein exhibits high affinity between biotin and rizavidin (dissociation constant [KD] ≈ 10- 15Utilizing the non-covalent bond of M), rizavidin is a biotin-binding protein that does not have significant predictive homology to human proteins. Rizavidin is a naturally occurring dimeric protein within the avidin protein family and was first discovered in Rhizobium etli, a symbiotic bacterium of kidney beans. Although rizavidin shares only 22% amino acid identity with chicken triavidin, a protein widely found in eggs, it exhibits high conservation of amino acid residues involved in biotin binding. No cross-reactivity to rizavidin has been observed in human serum samples obtained from subjects exposed to avidin [Helppolainen et al., 2007], suggesting that antibodies to rizavidin cannot cross-react with chicken triavidin. Biotin conjugates have been used in several clinical applications without reports of adverse events [Buller et al., 2014; Paty et al., 2010; Lazzeri et al., 2004].

[0177] In some embodiments, a biotin-binding site useful in accordance with this disclosure includes, for example, rizavidin or its biotin-binding domain or biotin-binding fragment, as further described in WO 2012 / 155053 (for the purposes described herein, this content is incorporated herein by reference in its entirety). In some embodiments, the biotin-binding site is or includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with rizavidin, or its biotin-binding domain or biotin-binding fragment. In some embodiments, a biotin-binding site useful in accordance with this disclosure includes the polypeptide of SEQ ID NO: 1 or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1, or its biotin-binding domain or biotin-binding fragment. In some embodiments, a biotin-binding site useful in accordance with this disclosure includes the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or its biotin-binding domain or biotin-binding fragment. In some embodiments, the biotin-binding site is or includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or its biotin-binding domain or biotin-binding fragment.

[0178] Fusion protein SPP2 In some embodiments, the fusion protein described herein is or comprises a complementary affinity molecule described herein (e.g., a biotin-binding site described herein) and one or more polypeptides of or derived from Streptococcus pneumoniae. In some embodiments, the fusion protein provided is SPP2, which is further described in International Application PCT / US2022 / 043156 filed on September 9, 2022 (for the purposes described herein, the contents thereof are incorporated herein by reference in their entirety). In some embodiments, the fusion protein comprises (i) a biotin-binding site (e.g., a biotin-binding protein), (ii) a non-hemolytic pneumolysin polypeptide or antigenic fragment thereof containing mutations in amino acid residues 294, 385, 428, and 433 of wild-type Streptococcus pneumoniae pneumolysin, and (iii) a Streptococcus pneumoniae elongation factor P(SP0435) polypeptide or antigenic fragment thereof. In some embodiments, the biotin-binding site is or includes a rizavidin polypeptide or its biotin-binding portion. In some embodiments, the fusion protein includes (i) a complementary affinity molecule as described herein (e.g., a biotin binding site as described herein), (ii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 7 (amino acids 2-470 of Streptococcus pneumoniae PdT(G294P)), and (iii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 9 (amino acids 62-185 of Streptococcus pneumoniae SP0435).In some embodiments, the fusion protein comprises a polypeptide containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein is (i) a complementary affinity molecule as described herein (e.g., a biotin-binding site as described herein), (ii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with pneumolysin, and (iii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SP0435. In some embodiments, the fusion protein is (i) a complementary affinity molecule as described herein (e.g., a biotin-binding site as described herein), (ii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with nonhemolytic pneumolysin containing mutations at amino acid residues 294, 385, 428, and 433 of wild-type Streptococcus pneumoniae pneumolysin, and (iii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SP0435.

[0179] In some embodiments, the fusion proteins described herein include (i) a complementary affinity molecule described herein (e.g., a biotin-binding site described herein) and (ii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of Sequence ID No. 7 (amino acids 2-470 of Streptococcus pneumoniae PdT(G294P)). In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with (i) a complementary affinity molecule as described herein (e.g., a biotin-binding site as described herein), and (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of Sequence ID No. 9 (amino acids 62-185 of Streptococcus pneumoniae SP0435).

[0180] In some embodiments, the fusion protein described herein includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof. In some embodiments, the fusion protein includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of Rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 7 (amino acids 2-470 of Streptococcus pneumoniae PdT(G294P)). In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 9 (amino acids 62-185 of Streptococcus pneumoniae SP0435).

[0181] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or less identity with the sequence of SEQ ID NO: 7 (Amino acids 2-470 of Streptococcus pneumoniae PdT (G294P)). (iii) a polypeptide or antigenic variant or fragment thereof comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, and (iii) a polypeptide or antigenic variant or fragment thereof 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 Streptococcus pneumoniae SP0435). In some embodiments, such a fusion protein further comprises one or more linkers.

[0182] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment site thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, or less identity with the sequence of SEQ ID NO: 7 (amino acids 2-470 of Streptococcus pneumoniae PdT (G294P)). (iii) a polypeptide or antigenic variant or fragment thereof comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, and (iii) a polypeptide or antigenic variant or fragment thereof 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 Streptococcus pneumoniae SP0435). In some embodiments, such a fusion protein further comprises one or more linkers.

[0183] In some embodiments, the fusion protein described herein is or includes an SPP2 fusion protein. In some embodiments, the SPP2 fusion protein comprises, from the N-terminus to the C-terminus, (i) a biotin-binding site (e.g., a biotin-binding protein), (ii) a first peptide linker, (iii) a pneumolysin (Ply) polypeptide or its antigenic variant or fragment described herein, (iv) a second peptide linker, and (v) an SP0435 polypeptide or its antigenic variant or fragment described herein. In such some embodiments, the biotin-binding site is or includes rizavidin or its biotin-binding portion. In some embodiments, the SPP2 fusion protein comprises (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin) or SEQ ID NO: 2 (amino acids 45-179 of Rizavidin) or SEQ ID NO: 3, or a biotin-binding site or biotin-binding fragment thereof; (ii) a first polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) a pneumolysin (Ply) polypeptide as described herein; (iv) a second polypeptide linker comprising the amino acid sequence of GGGGSSS (SEQ ID NO: 38); and (v) an SP0435 polypeptide as described herein. In some embodiments, the SPP2 fusion protein comprises, from the N-terminus to the C-terminus, (i) a biotin-binding site or biotin-binding fragment thereof that is or contains the polypeptide of SEQ ID NO: 1 (rizavidin), SEQ ID NO: 2 (amino acids 45-179 of rizavidin), or SEQ ID NO: 3; (ii) a first polypeptide linker containing the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) a pneumolysin (Ply) polypeptide as described herein; (iv) a second polypeptide linker containing the amino acid sequence of GGGGSSS (SEQ ID NO: 38); and (v) an SP0435 polypeptide as described herein.In some embodiments, such an SPP2 fusion protein may further include a detection or purification tag, such as a His tag. In some embodiments, an SPP2 fusion protein including a His tag is referred to as SPP2-H. In some such embodiments, the SPP2 fusion protein includes an SP0435 polypeptide (e.g., as described herein) between the biotin binding site and the Ply polypeptide (e.g., as described herein). In some embodiments, the SPP2 fusion protein may include a Ply polypeptide between the biotin binding site and the SP0435 polypeptide. In some embodiments, the Ply polypeptide contained in the fusion protein described herein is or includes an amino acid sequence or antigenic variant or fragment thereof that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of Sequence ID No. 7 (amino acids 2-470 of Streptococcus pneumoniae PdT (G294P)). In some embodiments, the SP0435 polypeptide contained in the fusion protein described herein is an amino acid sequence or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 9 (amino acids 62-185 of Streptococcus pneumoniae SP0435), or includes an antigenic variant or fragment thereof. In some embodiments, the fusion protein described herein 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 with the sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein described herein includes the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein described herein consists of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0184] Fusion protein CP1 In some embodiments, the fusion protein described herein is or comprises a complementary affinity molecule described herein (e.g., a biotin-binding site described herein) and one or more polypeptides of or derived from Streptococcus pneumoniae. In some embodiments, the fusion protein described herein is CP1, which is further described in International Patent Publication WO 2020 / 056127 (for the purposes described herein, the contents of which are incorporated herein in their entirety by reference). In some embodiments, the fusion protein comprises (i) a complementary affinity molecule as described herein (e.g., a biotin binding site as described herein), (ii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785), and (iii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500). In some embodiments, the fusion protein comprises a polypeptide containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.In some embodiments, the fusion protein is (i) a complementary affinity molecule as described herein (e.g., a biotin-binding site as described herein), (ii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SP0785, and (iii) a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SP1500.

[0185] In some embodiments, the fusion proteins described herein include (i) a polypeptide or antigenic variant or fragment thereof having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the complementary affinity molecule described herein (e.g., a biotin binding site described herein) and the sequence of Sequence ID No. 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785). In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with (i) a complementary affinity molecule as described herein (e.g., a biotin binding site as described herein), and (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of Sequence ID No. 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500).

[0186] In some embodiments, the fusion protein described herein includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof. In some embodiments, the fusion protein includes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of Rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785). In some embodiments, the fusion protein comprises a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500).

[0187] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785); (iii) a polypeptide or antigenic variant or fragment thereof comprising an amino acid sequence having 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, and (iii) a polypeptide or antigenic variant or fragment thereof 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 Streptococcus pneumoniae SP1500). In some embodiments, such a fusion protein further comprises one or more linkers.

[0188] In some embodiments, the fusion proteins described herein include each of the following: (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2 (amino acids 45-179 of rizavidin) or SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a polypeptide having at least 80%, at least 85%, at least 90%, or less identity with the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785). (iii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500), and (iii) a polypeptide or antigenic variant or fragment thereof containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500). In some embodiments, such a fusion protein further comprises one or more linkers.

[0189] In some embodiments, the fusion protein described herein is or includes a CP1 fusion protein. In some embodiments, the CP1 fusion protein comprises, from the N-terminus to the C-terminus, (i) a biotin-binding site (e.g., a biotin-binding protein), (ii) a first peptide linker, (iii) an SP1500 polypeptide or its antigenic variant or fragment as described herein, (iv) a second peptide linker, and (v) an SP0785 polypeptide or its antigenic variant or fragment as described herein. In such some embodiments, the biotin-binding site is or includes rizavidin or its biotin-binding portion. In some embodiments, the CP1 fusion protein comprises (i) a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1 (Rizavidin), or the sequence of SEQ ID NO: 2 (amino acids 45-179 of Rizavidin), or the sequence of SEQ ID NO: 3, or a biotin-binding site containing the same, or a biotin-binding fragment thereof; (ii) a first polypeptide linker containing the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) the SP0785 polypeptide as described herein; (iv) a second polypeptide linker containing the amino acid sequence of AAA; and (v) the SP1500 polypeptide as described herein. In some embodiments, the CP1 fusion protein comprises, from the N-terminus to the C-terminus, (i) a biotin-binding site or biotin-binding fragment thereof that is or contains the polypeptide of SEQ ID NO: 1 (rizavidin), SEQ ID NO: 2 (amino acids 45-179 of rizavidin), or SEQ ID NO: 3; (ii) a first polypeptide linker containing the amino acid sequence of GGGGSSS (SEQ ID NO: 38); (iii) the SP1500 polypeptide as described herein; (iv) a second polypeptide linker containing the amino acid sequence of AAA; and (v) the SP0785 polypeptide as described herein. In some embodiments, such a CP1 fusion protein may further include a detection or purification tag, such as a His tag.In some such embodiments, the CP1 fusion protein contains the SP1500 polypeptide between the biotin-binding site and the SP0785 polypeptide. In some embodiments, the CP1 fusion protein may contain the SP0785 polypeptide between the biotin-binding site and the SP1500 polypeptide. In some embodiments, the SP0785 polypeptide contained in the fusion protein described herein is or contains an amino acid sequence or antigenic variant or fragment thereof that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the sequence of SEQ ID NO: 11 (amino acids 33-399 of Streptococcus pneumoniae SP0785). In some embodiments, the SP1500 polypeptide contained in the fusion protein described herein is an amino acid sequence or antigenic variant or fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the sequence of SEQ ID NO: 13 (amino acids 27-278 of Streptococcus pneumoniae SP1500), or includes an antigenic variant or fragment thereof. In some embodiments, the fusion protein 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 the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0190] polymer In some embodiments, the immunogenic complexes described herein (e.g., MAPS complexes) include polymers. In some embodiments, the components of MAPS are polymers or include polymers. Polymers may be antigenic or non-antigenic. Polymers may be made from a wide range of materials. In some embodiments, the polymers are synthetic polymers. In some embodiments, the polymers are naturally occurring polymers. In some embodiments, the polymers are polysaccharides. In some embodiments, the polysaccharides are antigenic polysaccharides (e.g., antigenic polysaccharides described herein). In some embodiments, the polysaccharides are bacterial cells, eukaryotic cells (e.g., fungal, insect, or plant cells) or derived therefrom. In some embodiments, the polymers are mammalian cells (e.g., virus-infected cells or cancer cells) or derived therefrom. Such polymers are known in the Art and are included in their use in methods and compositions such as those disclosed herein.

[0191] In some embodiments, the polymer is a chimeric polymer comprising one or more types of polymers. For example, the polymer of an immunogenic complex as disclosed herein may comprise a first polymer or a first portion derived therefrom, and a second polymer or a second portion derived therefrom. There is no limit to the amount of different types of polymers that can be used in a single immunogenic complex. In some embodiments, the polymer is a single-chain polymer. In some embodiments, the polymer is a branched polymer comprising a chain polymer and one or more branched polymers. The chain polymer may be the first polymer or derived therefrom. The branched polymer may be at least one, at least two, at least three, or more different polymers or derived therefrom.

[0192] In some embodiments, the 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 repeating hydrocarbon 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.

[0193] In some embodiments, the polymer is a high molecular weight polymer, for example, the polymer may have an average molecular weight of about 425 to about 500 kDa, e.g., 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 is typically less than 500 kDa.

[0194] In some embodiments, the polymer is a low molecular weight polymer, for example, the polymer may have an average molecular weight of 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.

[0195] In some embodiments, the polymer (e.g., polysaccharide) is recovered from a natural source and / or purified therefrom, and in other embodiments, the polymer is a synthetic product. Methods for producing synthetic polymers (including synthetic polysaccharides) are known to those of the art and are encompassed in compositions and methods such as those disclosed herein.

[0196] In some embodiments, polymers that can be used in immunogenic complexes (e.g., MAPS complexes) described herein include dextran, polyethylene glycol polymers, poly(orthoester) polymers, polyacrylic carriers, PLGA, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimers, β-aminoester polymers, polyphosphoesters (PPE), liposomes, polymerosomes, nucleic acids, polyphosphorothioated oligonucleotides, chitosan, silk, polymer micelles, protein polymers, virus particles, virus-like particles (VLPs), or other microparticles. See, for example, 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 suitable for use as polymers herein. For example, see Biocompatible Pol. Nucl. Acid. Deliv. (Domb et al., eds., John Wiley & Sons, Inc. Hoboken, NJ, 2011).

[0197] For example, VLPs are similar to viruses, but are non-infectious because they do not contain viral genetic material. Expression (including recombinant expression) of viral structural proteins, such as envelope or capsid components, can lead to the self-assembly of VLPs. VLPs are made from components of a wide range of viral families, including Parvoviridae (e.g., adeno-associated viruses), Retroviridae (e.g., HIV), and Flaviviridae (e.g., hepatitis B virus or hepatitis C virus). VLPs can be produced in various cell culture systems, including mammalian cell lines, insect cell lines, yeast, and plant cells. Recombinant VLPs are particularly advantageous because their viral components can be fused to recombinant antigens, as described herein.

[0198] antigenic polysaccharide In some embodiments, the polymer used in the immunogenic complex is or contains an antigenic polysaccharide. In some embodiments, the antigenic polysaccharide is derived from an organism selected from the group consisting of bacteria, archaea, viruses, or eukaryotic cells such as fungi, insects, plants, or chimeras thereof. In some embodiments, the antigenic polysaccharide is a polysaccharide found to be present in or on mammalian cells, for example, cancer cells in some embodiments. In some embodiments, the antigenic polysaccharide is derived from pathogenic bacteria or viruses. In some embodiments, the antigenic polysaccharide is or contains an antigenic polysaccharide associated with a pathogen (e.g., bacteria, fungi, parasites, or viruses). In some embodiments, the antigenic polysaccharide is or contains an antigenic polysaccharide associated with a tumor. In some embodiments, the antigenic polysaccharide is or contains a glycoprotein. In certain embodiments, the antigenic polysaccharide is the capsular polysaccharide of Streptococcus pneumoniae, the cell wall polysaccharide of Streptococcus pneumoniae, the polysaccharide of Neisseria meningitidis, the polysaccharide of Haemophilus influenzae type b, the polysaccharide of Streptococcus agalactiae, the polysaccharide of Salmonella typhi Vi, the polysaccharide of Klebsiella, the polysaccharide of Pseudomonas, the polysaccharide of Escherichia coli, or the polysaccharide of Staphylococcus aureus. In some embodiments, the antigenic polysaccharide is derived from a tumor.

[0199] In some embodiments, the antigenic polysaccharide is or is derived from Gram-negative bacteria and / or Gram-positive bacteria. In some embodiments, the antigenic polysaccharide is or is derived from one or more glycoproteins. In some embodiments, one or more such glycoproteins is or is derived from one or more viruses. In some embodiments, the antigenic polysaccharide is or is derived from Streptococcus pneumoniae. In some embodiments, the antigenic polysaccharide contained in the immunogenic compositions described herein is or is derived from one or more pathogens. In some embodiments, one or more antigenic polysaccharides is or is derived from serotypes or strains (variants) of pathogens 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. In some embodiments, one or more antigenic polysaccharides are or are derived from more than 25 serotypes or strains (variants) of the pathogen, for example, serotypes or strains 26, 27, 28, 29, 30, 35, 40, 45, or 50. 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 the pathogen.

[0200] In some embodiments, the antigenic polysaccharide may be a branched polysaccharide, or alternatively, a linear polysaccharide.

[0201] In some embodiments, the antigenic polysaccharide is Vi antigen (capsular polysaccharide of Salmonella typhus), capsular polysaccharide of Streptococcus pneumoniae, cell wall polysaccharide of Streptococcus pneumoniae, capsular polysaccharide of Hib (Haemophilus influenzae type B), capsular polysaccharide of Neisseria meningitidis, Bacillus anthrax polysaccharide (the causative agent of anthrax), and polysaccharides of the capsule or cell wall of other bacteria, or any combination thereof.

[0202] In some embodiments, the antigenic polysaccharide consists of or contains a sugar moiety. For example, in some embodiments, the polysaccharide is the Vi polysaccharide of salmonella typhus. Vi capsular polysaccharides have been developed for 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 α-1→4 galacturonic acid having an N-acetyl group at the C-2 position and a variable O-acetyl group at the C-3 position. The toxicity of S. typhus correlates with the expression of this molecule. Sharma et al., 101 PNAS 17492 (2004). Vi polysaccharide vaccines for salmonella typhus have several advantages, including infrequent and mild side effects, and consistent immunogenicity and efficacy with a single dose. Vi polysaccharide can be reliably standardized by physicochemical methods validated with other polysaccharide vaccines, and Vi is stable at room temperature and can be administered concurrently with other vaccines without affecting immunogenicity and tolerance. Azze et al., 21 Vaccine 2758 (2003).

[0203] In some embodiments, the antigenic polysaccharide may be derived from a capsular polysaccharide of Neisseria meningitidis, for example, serogroup A, C, W, W135, or Y, at least 1, 2, 3, or 4. In some embodiments, the polysaccharide includes type 5, type 8, or any of these polysaccharides or oligosaccharides of Staphylococcus aureus.

[0204] In some embodiments, the antigenic polysaccharide is derived from Klebsiella pneumoniae and may be, for example, a lipopolysaccharide (LPS)-derived polysaccharide or a capsular polysaccharide. In some embodiments, the LPS-derived polysaccharide is O-polysaccharide (OPS). In some embodiments, the LPS-derived polysaccharide is core O-polysaccharide (COPS). In some embodiments, the polysaccharide is from or derived from OPS of Klebsiella pneumoniae serotype O1, O2, O2ac, O3, O4, O5, O7, O8, or O12. In some embodiments, the polysaccharide is from or derived from CPS of Klebsiella pneumoniae K1, K2, K10, K16, or K19.

[0205] In some embodiments, the antigenic polysaccharide is from or derived from Pseudomonas aeruginosa, for example, OPS, LPS, or an extracellular polysaccharide. In some embodiments, the antigenic polysaccharide is from or derived from OPS of Pseudomonas aeruginosa selected from serotypes O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, and O20. In some embodiments, the antigenic polysaccharide is a capsule or capsule-like polysaccharide derived from alginate, PsL, or Pel of Pseudomonas aeruginosa, or derived therefrom. In some embodiments, the antigenic polysaccharide is an extracellular polysaccharide derived from PsL of Pseudomonas aeruginosa, or derived therefrom.

[0206] In some embodiments, the antigenic polysaccharides present in the immunogenic complex described herein include one or more polysaccharides derived from tumors.

[0207] In some embodiments, the antigenic polysaccharide is from or derived from *Streptococcus pneumoniae* polysaccharide. In some embodiments, the antigenic polysaccharide present in the immunogenic complex described herein includes one *Streptococcus pneumoniae* polysaccharide. Capsular polysaccharides are used to distinguish serotypes of *Streptococcus pneumoniae*. There are at least 97 different serotypes of *Streptococcus pneumoniae* polysaccharide, each with a different chemical structure. Figure 6 illustrates exemplary structural and chemical information of the capsular polysaccharide of a specific *Streptococcus pneumoniae*. All structures are derived from the European Encyclopedia of Pharmaceutical Sciences 9.0. Serotype names used herein are in accordance with Danish nomenclature (Kauffmann et al., Intl. Bull. Bact. Nomenclature and Taxonomy 10:31-41 (1960); Geno et al., Clin Microbiol Rev 28(3):871-899 (2015)).

[0208] In some embodiments, the antigenic polysaccharides present in the immunogenic complex are 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, 1 1A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 2 It contains one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 0B, 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.

[0209] In some embodiments, the antigenic polysaccharides present in the immunogenic complex include one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae 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.

[0210] In some embodiments, the antigenic polysaccharides present in the immunogenic complex include one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 6C, 7C, 15A, 16F, 23A, 23B, 24F, 31, 35B, and 38.

[0211] Exemplary methods for isolating and purifying polysaccharides In some embodiments, the disclosure provides a method for purifying one or more polysaccharides described herein from one or more cellular components of a bacterium. In some embodiments, the method includes purifying capsular polysaccharides from one or more cellular components of a bacterium.

[0212] In some embodiments, the bacteria are Gram-negative. In some embodiments, the bacteria are Gram-positive. In some embodiments, the bacteria are Streptococcus pneumoniae.

[0213] In some embodiments, the cellular components include proteins. In some embodiments, the cellular proteins include nucleic acids. 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 the lysis solution.

[0214] In some embodiments, the polysaccharide purification process incorporates a series of ethanol precipitation steps, washing the crude polysaccharide preparation with ethanol, diethyl ether, and / or acetone, and drying under vacuum to yield a purified product. In some embodiments, a phenol extraction step is incorporated for polysaccharide purification. In some embodiments, the purification process utilizes a CTAB (cetyltrimethylammonium bromide) precipitation step in addition to the use of ethanol precipitation and phenol precipitation steps.

[0215] Exemplary methods of biotinylation of polysaccharides In some embodiments, the present disclosure provides a method of biotinylating one or more polysaccharides described herein. In some embodiments, the method comprises reacting a purified polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to activate the hydroxyl groups of the polysaccharide, followed by adding amine PEG biotin under conditions that result in a covalent linkage between biotin and the polysaccharide. In some embodiments, the desired biotinylation level is achieved by varying the ratio of CDAP to the polysaccharide. In some embodiments, the biotinylated polysaccharide is purified by filtration to remove process residues such as unreacted biotin, dimethylaminopyridine, acetonitrile, cyanide, and unreacted glycine. In some embodiments, the biotinylation level of the polysaccharides described herein is optimized such that the amount of biotin available after MAPS conjugation is reduced.

[0216] Immunogenic conjugate The techniques and methods described herein (e.g., the use of aluminum-based adjuvants at low concentrations) are also applicable to conjugate vaccines. In some embodiments, the conjugate vaccine is or comprises an immunogenic conjugate. As described herein, the immunogenic conjugate comprises (ii) one or more polypeptides (e.g., antigenic polypeptides) as described herein, conjugated to one or more polymers (e.g., antigenic polysaccharides) as described herein. In some embodiments, the one or more polypeptides (e.g., antigenic polypeptides) as described herein are covalently bonded to one or more polysaccharides as described herein. In some embodiments, the one or more polypeptides of the immunogenic conjugate comprise a carrier protein (e.g., one of those described herein).

[0217] In some embodiments, one or more polysaccharides of the immunogenic conjugate include the capsular polysaccharide of Streptococcus pneumoniae. In some embodiments, one or more polypeptides of the immunogenic conjugate include the antigenic polypeptide of Streptococcus pneumoniae. In some embodiments, the antigenic polypeptide of the immunogenic conjugate is or includes a fusion protein. In some such embodiments, the fusion protein of the immunogenic conjugate is or includes an SPP2 fusion protein. In some such embodiments, the fusion protein of the immunogenic conjugate is or includes a CP1 fusion protein. In some embodiments, the antigenic polypeptide or fusion protein of the immunogenic conjugate is or includes a carrier protein.

[0218] In some embodiments, the immunogenic conjugate comprises one or more capsular polysaccharides of Streptococcus pneumoniae selected from or derived from one or more serotypes of Streptococcus pneumoniae including 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 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. In some embodiments, the immunogenic conjugate comprises one or more capsular polysaccharides of Streptococcus pneumoniae selected from or derived from one or more serotypes of Streptococcus pneumoniae including 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 1OA, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 38.

[0219] In some embodiments, the immunogenic conjugate is (a) one or more carrier proteins described herein, and (b)1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N , 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, It comprises one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 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 (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, 34, or more), Here, one or more carrier proteins are covalently attached to one or more polysaccharides.

[0220] In some embodiments, the immunogenic conjugate is (a) one or more carrier proteins described herein, and (b) comprising one or more serotypes of Streptococcus pneumoniae selected from or derived 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, and 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) of Streptococcus pneumoniae, Here, one or more carrier proteins are covalently attached to one or more polysaccharides.

[0221] In some embodiments, the immunogenic conjugate is (a) one or more SPP2 proteins, and (b)1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N , 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, It comprises one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 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 (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, 34, or more), Here, one or more SPP2 proteins are covalently attached to one or more polysaccharides.

[0222] In some embodiments, the immunogenic conjugate is (a) one or more SPP2 proteins, and (b) comprising one or more serotypes of Streptococcus pneumoniae selected from or derived 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, and 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) of Streptococcus pneumoniae, Here, one or more SPP2 proteins are covalently attached to one or more polysaccharides.

[0223] In some embodiments, the immunogenic conjugate is (a) one or more CP1 proteins, and (b)1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N , 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, It comprises one or more Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 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 (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33, 34, or more), Here, one or more CP1 proteins are covalently attached to one or more polysaccharides.

[0224] In some embodiments, the immunogenic conjugate is (a) one or more CP1 proteins, and (b) comprising one or more serotypes of Streptococcus pneumoniae selected from or derived 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, and 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) of Streptococcus pneumoniae, Here, one or more CP1 proteins are covalently attached to one or more polysaccharides.

[0225] In some embodiments, the immunogenic composition includes one or more immunogenic conjugates: (a) one or more SPP2 proteins, and (b)1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12 F,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, one or more primary Streptococcus pneumoniae capsular polysaccharides from or derived therefrom, wherein one or more SPP2 proteins are covalently attached to one or more primary polysaccharides, or (c) One or more CP1 proteins, and (d)1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 1 2F, 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, One or more secondary Streptococcus pneumoniae capsular polysaccharides from or derived from one or more serotypes of Streptococcus pneumoniae selected from 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 one or more CP1 proteins are covalently attached to the one or more secondary polysaccharides.

[0226] Manufacturing of immunogenic complexes and immunogenic conjugates This disclosure includes methods for producing immunogenic complexes as described herein. In some embodiments, the method for producing an immunogenic complex includes the step of complexing at least one biotinylated polymer (e.g., biotinylated polysaccharide (e.g., biotinylated polysaccharide as described herein)) with at least one biotin-binding fusion protein as described herein. In some embodiments, this disclosure includes methods for producing immunogenic conjugates as described herein. In some embodiments, the method for producing an immunogenic conjugate includes the step of conjugating at least one polymer (e.g., polysaccharide (e.g., antigenic polysaccharide)) with at least one polypeptide (e.g., antigenic polypeptide) as described herein.

[0227] In some embodiments, the average ratio (e.g., mean) of protein (e.g., antigenic protein) to polymer (e.g., polysaccharide) of multiple immunogenic complexes or 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 ratio of protein (e.g., polysaccharide) to polymer (e.g., polysaccharide) of multiple immunogenic complexes or conjugates is approximately 1:1 (w / w). In some embodiments, the average ratio of protein (e.g., polysaccharide) to polymer (e.g., polysaccharide) of multiple immunogenic complexes or conjugates is approximately 2:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or conjugates is approximately 3:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or conjugates is approximately 4:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or conjugates is approximately 5:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or conjugates is approximately 6:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or conjugates is approximately 7:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or immunogenic conjugates is approximately 8:1 (w / w). In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or immunogenic conjugates is approximately 9:1 (w / w).In some embodiments, the average ratio of protein to polymer (e.g., polysaccharide) in multiple immunogenic complexes or immunogenic conjugates is approximately 10:1 (w / w). The immunogenic compositions and vaccines of the present invention may comprise mixtures of immunogenic complexes or immunogenic conjugates with different average ratios of protein to polymer (e.g., polysaccharide).

[0228] In some embodiments, the immunogenic composition (e.g., a vaccine) comprises a plurality of immunogenic complexes comprising the fusion protein and capsular polysaccharide described herein. 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 multiple immunogenic complexes is approximately 3:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 5:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 6:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 8:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 9:1 (w / w). In some embodiments, the average ratio of fusion proteins to capsular polysaccharides in multiple immunogenic complexes is approximately 10:1 (w / w).In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is selected to enhance the immunogenic potential (carrier or presentation function) of the polysaccharide and / or to induce defense against pathogens or tumors through a protein-specific immune response, or to suppress pathogens or tumors. The immunogenic compositions of this disclosure (e.g., vaccines) may comprise mixtures of immunogenic complexes with different average ratios of protein to polysaccharide.

[0229] In some embodiments, the method for producing an immunogenic complex includes the step of complexing at least one biotinylated polysaccharide with at least one biotin-binding fusion protein. In some embodiments, the fusion protein contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, the fusion protein contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with SEQ ID NO: 30 or SEQ ID NO: 31.

[0230] In some embodiments, the immunogenic composition (e.g., vaccine) comprises a plurality of immunogenic complexes comprising a fusion protein and a capsular polysaccharide. In some embodiments, the fusion protein is the SPP2 protein. In some embodiments, the fusion protein is the CP1 protein. In some embodiments, the capsular polysaccharide is from or derived from Streptococcus 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 Streptococcus pneumoniae having a serotype selected from the group including serotypes 30, 31, 32, or 33 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 average ratio of fusion protein to capsular polysaccharide in multiple 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 multiple immunogenic complexes is approximately 1:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 2:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 3:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 5:1 (w / w).In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 6:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 7:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 8:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 9:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 10:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is selected to enhance the immunogenic potential (carrier function) of the polysaccharide and / or to induce defense against colonization of pneumococci by any pneumococcus (regardless of the polysaccharide serotype) or to suppress colonization of pneumococci by any pneumococcus (regardless of the polysaccharide serotype) through a protein-specific immune response. The immunogenic compositions of this disclosure (e.g., vaccines) may comprise mixtures of immunogenic complexes with different average ratios of protein to polysaccharide.

[0231] In some embodiments, the 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 the CP1 protein. In some embodiments, the capsular polysaccharide is from or derived from Streptococcus 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 Streptococcus pneumoniae having a serotype selected from the group including serotypes 20, 21, 22, or 23, which are 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 average ratio of fusion protein to capsular polysaccharide in multiple 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 multiple immunogenic complexes is approximately 1:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 2:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 3:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 4:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 5:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 6:1 (w / w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in multiple immunogenic complexes is approximately 7:1 (w / w).In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the plurality of immunogenic complexes is approximately 8:1 (w / w). In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the plurality of immunogenic complexes is approximately 9:1 (w / w). In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the plurality of immunogenic complexes is approximately 10:1 (w / w). In some embodiments, the average ratio of the fusion protein to the capsular polysaccharide in the plurality of immunogenic complexes is selected to enhance the immunogenic potential (carrier function) of the polysaccharide and / or to elicit protection against pneumococcal colonization by any pneumococcus (regardless of the polysaccharide serotype) through a protein-specific immune response or to inhibit pneumococcal colonization by any pneumococcus (regardless of the polysaccharide serotype). The immunogenic compositions (e.g., vaccines) of the present disclosure may include mixtures of immunogenic complexes having different average ratios of protein to polysaccharide.

[0232] immunogenic composition Other aspects of this disclosure provide compositions comprising one or more immunogenic complexes described herein. For example, an immunogenic composition, such as a vaccine composition, may comprise one or more immunogenic complexes described herein. In some embodiments, such a composition may comprise a plurality of one type of immunogenic complex described herein. For example, a composition may comprise a population of one type of immunogenic complex, where all immunogenic complexes comprise the same polypeptide (e.g., antigenic polypeptide) and the same polymer (e.g., antigenic polysaccharide). Additionally or alternatively, such a composition may comprise more than one type of immunogenic complex described herein. For example, a composition may comprise a population of different types of immunogenic complexes. In some embodiments, a composition may comprise a population of a first type of immunogenic complex and a population of a second type of immunogenic complex, where the first and second types of immunogenic complexes comprise different polypeptides (e.g., antigenic polypeptide) and / or different polymers (e.g., antigenic polysaccharide). In some embodiments, the composition may comprise a group of first-order immunogenic complexes and a group of second-order immunogenic complexes, where the first and second-order immunogenic complexes comprise the same polypeptide (e.g., antigenic polypeptide) and different polymers (e.g., antigenic polysaccharides (e.g., polysaccharides of different serotypes)).

[0233] In some embodiments, the immunogenic composition is a monovalent immunogenic composition. In some embodiments, the immunogenic composition is a polyvalent or multivalent immunogenic composition. In some embodiments, the immunogenic composition is a monovalent immunogenic composition containing one or more antigens derived from one strain or variant of a pathogen. In some embodiments, the immunogenic composition is a multivariant immunogenic composition containing one or more antigens derived from more than one strain or variant of a pathogen. In some embodiments, the valence of the immunogenic composition refers to the number of types of immunogenic complexes present in the immunogenic composition. The immunogenic valence described herein is not limited to the total number of antigens present in the immunogenic composition or the number of pathogen strains for which administration of the immunogenic composition can induce an immune defense response. In non-limiting examples, a 34-valent immunogenic composition may contain more than 34 antigenic components (e.g., peptide and / or polysaccharide components) and may induce an immune defense response against more than 34 pathogens or serotypes or strains of pathogens.

[0234] In some embodiments, the immunogenic composition contains 1 to 60 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 50 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 45 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 40 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 35 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 34 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 33 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 32, 1 to 31, or 1 to 30 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 30 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 24 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 15 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 9 immunogenic complexes. In some embodiments, the immunogenic composition contains 1 to 5 immunogenic complexes. In some embodiments, the immunogenic composition is a polyvalent composition. In some embodiments, the immunogenic composition contains at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 immunogenic complexes. In some embodiments, the immunogenic composition contains at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 immunogenic complexes. In some embodiments, the immunogenic composition contains at least 30 (e.g., at least 31, at least 32, at least 33, at least 34) immunogenic complexes described herein.In some embodiments, the immunogenic composition comprises 30 to 40 immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 30 to 35 immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 25 to 50 or 25 to 40 immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 60 or fewer immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 50 or fewer immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 45 or fewer immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 40 or fewer immunogenic complexes described herein. In some embodiments, the immunogenic composition comprises 35 or fewer immunogenic complexes described herein.

[0235] In some embodiments, the immunogenic composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 immunogenic complexes described herein.

[0236] In some embodiments, the immunogenic composition contains two or more immunogenic complexes in such quantities that the weight of polymers (e.g., polysaccharides) in the immunogenic composition derived from each immunogenic complex is approximately the same, for example, in a w / w ratio of about 1:1. In some embodiments, the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by each immunogenic complex is about 0.20 μg, about 0.25 μg, about 0.5 μg, about 1 μg, about 1.5 μg, about 2 μg, about 2.5 μg, about 3 μg, about 3.5 μg, about 4 μg, about 4.5 μg, about 5 μg, about 5.5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, or about 12 μg. In some embodiments, the weight of the polymer (e.g., polysaccharide) in the immunogenic composition provided by each immunogenic complex is greater than 12 μg, for example, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg, or more.

[0237] In some embodiments, the immunogenic composition contains two or more immunogenic complexes in such quantities that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by each immunogenic complex is different, for example, not about 1:1 in w / w ratio. In some embodiments, the immunogenic composition contains two or more immunogenic complexes in such quantities that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by the first immunogenic complex and the 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 contains a mixture of immunogenic complexes such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by the immunogenic complexes is in the range of about 0.20 μg to about 6 μg. In some embodiments, the immunogenic composition contains a mixture of immunogenic complexes such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by the immunogenic complexes ranges from about 0.20 μg to about 12 μg. In some embodiments, the immunogenic composition contains a mixture of immunogenic complexes such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by each immunogenic complex ranges from about 0.20 μg to about 20 μg. In some embodiments, the immunogenic composition contains a mixture of immunogenic complexes such that the weight of polymers (e.g., polysaccharides) in the immunogenic composition provided by each immunogenic complex ranges from about 0.20 μg to about 40 μg.

[0238] In some embodiments, the immunogenic composition contains two or more immunogenic complexes in such quantities that the combined weight of the polymer (e.g., polysaccharide) and polypeptide in the immunogenic composition provided by each immunogenic complex is approximately the same, for example, with a protein:PS w / w ratio of about 1:1. In some embodiments, the immunogenic composition contains two or more immunogenic complexes in such quantities that the combined weight of the polymer (e.g., polysaccharide) and polypeptide in the immunogenic composition provided by each immunogenic complex is such that the protein:PS w / w ratio is 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.

[0239] In some embodiments, the combined weight of the polymer (e.g., polysaccharide) and polypeptide in the immunogenic composition provided by each immunogenic complex is approximately 0.20 μg, approximately 0.40 μg, approximately 1 μg, approximately 2 μg, approximately 3 μg, approximately 4 μg, approximately 5 μg, approximately 6 μg, approximately 7 μg, approximately 8 μg, approximately 9 μg, approximately 10 μg, approximately 11 μg, approximately 12 μg, approximately 14 μg, approximately 16 μg, approximately 18 μg, approximately 20 μg, approximately 21 μg, approximately 22 μg, approximately 23 μg, approximately 24 μg, approximately 25 μg, approximately 30 μg, approximately 40 μg, approximately 50 μg, approximately 60 μg, approximately 70 μg, approximately 80 μg, approximately 90 μg, approximately 100 μg, or approximately 110 μg.

[0240] In some embodiments, the immunogenic composition contains two or more immunogenic complexes (for example, in the immunogenic composition) in amounts such that the combined weight of the polymer (e.g., polysaccharide) and polypeptide in the immunogenic composition provided by each immunogenic complex differs, for example, in w / w, the protein:PS ratio is not about 1:1, for example, the protein:PS ratio is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the immunogenic composition contains a mixture of immunogenic complexes such that the combined weight of the polymer (e.g., polysaccharide) and polypeptide in the immunogenic composition provided by each immunogenic complex is in the range of about 0.4 μg to about 110 μg.

[0241] In some embodiments, the immunogenic compositions described herein include an adjuvant.

[0242] In some embodiments, the immunogenic compositions described herein are vaccine compositions.

[0243] In some embodiments, the immunogenic composition is an immunogenic composition described in WO 2020 / 056202 (for the purposes described herein, this content is incorporated herein in its entirety by reference). In some embodiments, the immunogenic composition is an immunogenic composition described in international application PCT / US2022 / 043156 (for the purposes described herein, this content is incorporated herein in its entirety by reference).

[0244] In some embodiments, the immunogenic composition includes one or more types of immunogenic complexes selected from the following (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more): (1) Biotinylated Streptococcus pneumoniae serotype 1 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (2) Biotinylated Streptococcus pneumoniae serotype 2 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (3) Biotinylated Streptococcus pneumoniae serotype 3 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (4) Biotinylated Streptococcus pneumoniae serotype 4 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (5) Biotinylated Streptococcus pneumoniae serotype 5 capsular polysaccharide, and an immunogenic complex containing one or more CP1 SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (6) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (7) Biotinylated Streptococcus pneumoniae serotype 6B capsular polysaccharide, and an immunogenic complex comprising one or more or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (8) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (9) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6D capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (10) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6E capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (11) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (12) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6G capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (13) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6H capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (14) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (15) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (16) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (17) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (18) Biotinylated Streptococcus pneumoniae serotype 8 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (19) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 9A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (20) Biotinylated capsular polysaccharide of Streptococcus pneumoniae serotype 9L, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (21) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 9N capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (22) Biotinylated Streptococcus pneumoniae serotype 9V capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (23) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 10A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (24) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 10B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (25) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 10C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (26) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 10F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (27) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (28) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (29) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (30) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11D capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (31) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11E capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (32) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (33) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 12A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (34) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 12B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (35) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 12F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (36) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 13 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (37) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 14 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (38) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 15A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (39) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 15B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (40) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 15C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (41) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 15F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (42) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 16A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (43) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 16F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (44) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 17A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (45) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 17F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (46) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 18A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (47) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 18B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (48) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 18C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (49) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 18F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (50) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 19A capsular polysaccharide and one or more CP1 SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (51) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 19B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (52) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 19C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (53) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 19F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (54) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 20A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (55) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 20B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (56) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 21 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (57) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 22A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (58) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 22F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (59) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 23A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (60) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 23B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (61) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 23F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (62) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 24A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (63) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 24B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (64) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 24F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (65) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 25A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (66) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 25F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (67) Biotinylated Streptococcus pneumoniae serotype 27 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (68) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 28A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (69) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 28F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (70) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 29 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (71) Biotinylated Streptococcus pneumoniae serotype 31 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (72) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 32A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (73) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 32F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (74) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 33A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (75) Biotinylated Streptococcus pneumoniae serotype 33B capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (76) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 33C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (77) Biotinylated Streptococcus pneumoniae serotype 33D capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (78) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 33E capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (79) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 33F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (80) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 34 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (81) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 35A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (82) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 35B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (83) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 35C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (84) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 35F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (85) Biotinylated Streptococcus pneumoniae serotype 36 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (86) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 37 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (87) Biotinylated Streptococcus pneumoniae serotype 38 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (88) Biotinylated Streptococcus pneumoniae serotype 39 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (89) Biotinylated Streptococcus pneumoniae serotype 40 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (90) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 41A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (91) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 41F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (92) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 42 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (93) Biotinylated Streptococcus pneumoniae serotype 43 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (94) Biotinylated Streptococcus pneumoniae serotype 44 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (95) Biotinylated Streptococcus pneumoniae serotype 45 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (96) Biotinylated Streptococcus pneumoniae serotype 46 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (97) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 47A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (98) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 47F capsular polysaccharide, and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (99) An immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 48 capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide.

[0245] In some embodiments, the immunogenic composition includes one or more types of immunogenic complexes selected from the following (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): (1) Biotinylated Streptococcus pneumoniae serotype 1 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (2) Biotinylated Streptococcus pneumoniae serotype 2 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (3) Biotinylated Streptococcus pneumoniae serotype 3 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (4) Biotinylated Streptococcus pneumoniae serotype 4 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (5) Biotinylated Streptococcus pneumoniae serotype 5 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (6) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide. (7) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6B capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (8) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 6C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (9) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7C capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (10) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 7F capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (11) Biotinylated Streptococcus pneumoniae serotype 8 capsular polysaccharide, and an immunogenic complex comprising one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (12) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 9N capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (13) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 9V capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (14) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 10A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (15) an immunogenic complex comprising biotinylated Streptococcus pneumoniae serotype 11A capsular polysaccharide and one or more CP1 or SPP2 fusion proteins non-covalently complexed with the biotinylated polysaccharide, (16) an immunogenic complex comprising biotinyl...

Claims

1. A pharmaceutical composition comprising an immunogenic composition containing one or more immunogenic complexes, At least one of the immunogenic complexes is (a) Biotinylated polysaccharide antigen, and (b) A fusion protein comprising (i) a biotin-binding site and (ii) at least one polypeptide antigen. Includes, The biotinylated polysaccharide antigen is non-covalently bound to the biotin-binding site of the fusion protein. And optionally, The pharmaceutical composition contains an aluminum-based adjuvant or an adjuvant containing the same, further comprising a low concentration of aluminum in the pharmaceutical composition. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises an aluminum-based adjuvant or an adjuvant containing the same, in a low concentration of aluminum.

3. A method for producing a pharmaceutical composition, The method includes the step of combining an immunogenic composition with an adjuvant, The immunogenic composition comprises one or more immunogenic complexes, At least one of the immunogenic complexes is (a) Biotinylated polysaccharide antigen, and (b) A fusion protein comprising (i) a biotin-binding site and (ii) at least one antigenic polypeptide. Includes, The biotinylated polysaccharide antigen is non-covalently bound to the biotin-binding site of the fusion protein, and The adjuvant is an aluminum-based adjuvant or is included in the pharmaceutical composition in the form of a low concentration of aluminum. method.

4. The pharmaceutical composition or method according to any one of claims 1 to 3, wherein the aluminum concentration in the pharmaceutical composition is lower than that of the reference composition.

5. The pharmaceutical composition, when administered to a subject, is characterized by inducing an immune response at a level higher than the corresponding reference level to (i) at least one of the polysaccharide antigens and / or (ii) at least one of the polypeptide antigens. Here, the reference level is the level of immune response induced in the subject after administration of the reference composition. A pharmaceutical composition or method according to any one of claims 1 to 4.

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

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

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

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

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

11. The pharmaceutical composition or method according to any one of claims 1 to 10, wherein the aluminum concentration in the pharmaceutical composition is about 0.125 mg / mL to about 1.25 mg / mL.

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

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

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

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

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

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

1.

18. The pharmaceutical composition or method according to any one of claims 1 to 17, wherein the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen 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 according to any one of claims 1 to 18, wherein the ratio of the mass of aluminum in the pharmaceutical composition to the total mass of the biotinylated polysaccharide antigen is about 1.91:1 to about 1.03:

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

33. The pharmaceutical composition or method according to any one of claims 1 to 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 according to claim 33, wherein the aluminum-based adjuvant is aluminum phosphate.

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

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

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

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

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

40. The pharmaceutical composition or method according to any one of claims 1 to 39, wherein the biotinylated polysaccharide antigen includes a bacterial polysaccharide antigen, a fungal polysaccharide antigen, a parasitic polysaccharide antigen, a viral polysaccharide antigen, and a mammalian polysaccharide antigen (for example, a tumor polysaccharide antigen).

41. The pharmaceutical composition or method according to claim 40, wherein the polysaccharide antigen is a polysaccharide selected from the group consisting of Salmonella polysaccharide, Streptococcus pneumoniae polysaccharide, Haemophilus polysaccharide, Neisseria meningitidis polysaccharide, Staphylococcus polysaccharide, Bacillus anthracis polysaccharide, Streptococcus polysaccharide, Pseudomonas polysaccharide, Klebsiella polysaccharide, Cryptococcus polysaccharide, polysaccharides of the capsule or cell wall of other bacteria, viral polysaccharides (e.g., viral glycoproteins), or a combination thereof.

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

43. The biotinylated polysaccharide antigen is 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E. , 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A pharmaceutical composition or method according to any one of claims 1 to 42, comprising one or more polysaccharides of Streptococcus pneumoniae, each having a serotype independently selected from 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.

44. The pharmaceutical composition or method according to any one of claims 1 to 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 according to any one of claims 1 to 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 according to any one of claims 1 to 45, wherein at least one of the polypeptide antigens is 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. At least one of the polypeptide antigens is a streptococcal antigen (e.g., Streptococcus pneumoniae, group A, group B, and viridans antigen), tuberculosis antigen, tetanus antigen, anthrax antigen, pertussis antigen, staphylococcal antigen (e.g., Staphylococcus aureus), Haemophilus antigen, Enterobacter antigen, Acinetobacter antigen, Citrobacter antigen, Serratia antigen, Clostridium antigen, Campylobacter antigen, Vibrio cholerae antigen, Pseudomonas antigen, Neisseria meningitidis antigen, Neisseria gonorrhoeae antigen, A pharmaceutical composition or method according to any one of claims 1 to 46, comprising an antigen selected from the group consisting of Chlamydia trachomatis antigen, Klebsiella antigen, Shigella antigen, Salmonella antigen, Escherichia coli antigen, malaria antigen, HIV antigen, HPV antigen, influenza (e.g., seasonal or epidemic) antigen, coronavirus antigen (e.g., SARS-CoV-2 antigen), herpes (e.g., HSV) antigen, tumor antigen, and a combination thereof.

48. A pharmaceutical composition or method according to any one of claims 1 to 47, wherein at least one of the polypeptide antigens is or contains a pneumococcal polypeptide antigen.

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

50. At least one of the immunogenic complexes is (i) Biotin binding site, (ii) pneumolysin polypeptide antigen or its antigenic fragment, (iii) SP0435 polypeptide antigen or its antigenic fragment A pharmaceutical composition or method according to any one of claims 1 to 49, comprising a fusion protein containing the above.

51. The pharmaceutical composition or method according to claim 50, wherein the fusion protein has an amino acid sequence that is 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, or contains the same.

52. At least one of the immunogenic complexes is (i) Biotin binding site, (ii) SP1500 polypeptide antigen or its antigenic fragment, and (iii) SP0785 polypeptide antigen or its antigenic fragment A pharmaceutical composition or method according to any one of claims 1 to 51, comprising a fusion protein containing the above.

53. The pharmaceutical composition or method according to claim 52, wherein the fusion protein has or contains an amino acid sequence that is 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 according to any one of claims 1 to 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 immunogenic complexes.

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

56. The pharmaceutical composition or method according to any one of claims 1 to 55, wherein one or more of the immunogenic complexes include the following: A first multi-type immunogenic complex comprising biotinylated polysaccharide antigens selected from serotypes 1, 4, 6A, 6B, 9V, 15B, 18C, 19A, 23F, and 33F of Streptococcus pneumoniae, wherein each biotinylated polysaccharide antigen is non-covalently complexed with the biotin-binding site of the first fusion protein: Here, the first fusion protein is, (a) Biotin binding site, (b) A first polypeptide antigen or antigenic fragment thereof containing an amino acid sequence identical to 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% of SEQ ID NO: 7, and (c) Sequence ID 9 contains a second polypeptide antigen or antigenic fragment thereof that has an identical amino acid sequence by 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%, and, A second multi-type immunogenic complex comprising biotinylated polysaccharide antigens selected from 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 of Streptococcus pneumoniae, wherein each biotinylated polysaccharide antigen is non-covalently complexed with the biotin-binding site of the second fusion protein: Here, the second fusion protein is (a) Biotin binding site, (b) A first polypeptide antigen or antigenic fragment thereof containing 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 amino acid sequence to SEQ ID NO: 13, and (c) SEQ ID NO: 11 contains a second polypeptide antigen or antigenic fragment thereof that has an identical amino acid sequence by 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%.

57. The pharmaceutical composition or method according to any one of claims 1 to 56, wherein the biotin-binding site is or comprises a rizavidin polypeptide.

58. The rizavidin polypeptide described above (i) A polypeptide or biotin-bound fragment thereof containing 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 amino acid sequence to SEQ ID NO: 1; or (ii) A polypeptide or biotin-bound fragment thereof containing an amino acid sequence identical to 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% of SEQ ID NO: 2 or SEQ ID NO:

3. The pharmaceutical composition or method according to any one of claims 1 to 57.

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

60. The aforementioned pharmaceutical composition (a) Buffer, optionally, wherein the buffer is a histidine buffer, succinate buffer, and / or phosphate buffer, (b) Salt, optionally, where salt is sodium chloride or contains sodium chloride. (c) Surfactants, optionally, wherein the surfactant is or contains polysorbate 80, and (d) Stabilizer, optionally, wherein the stabilizer is or contains sucrose. A pharmaceutical composition or method according to any one of claims 1 to 59, further comprising one or more of the above.

61. The pharmaceutical composition or method according to any one of claims 1 to 60, wherein the pharmaceutical composition further comprises a succinate buffer or a buffer containing the same.

62. A dosage form of the pharmaceutical composition according to any one of claims 1, 2, and 4 to 61.

63. The dosage form according to claim 62, wherein the amount of aluminum in the dosage form is less than 0.625 mg.

64. The dosage form according to claim 62 or claim 63, wherein the amount of aluminum in the dosage form is approximately 0.325 mg to approximately 0.175 mg.

65. The dosage form according to any one of claims 62 to 64, wherein the amount of aluminum in the dosage form is approximately 0.25 mg.

66. The dosage form according to any one of claims 62 to 65, wherein the amount of aluminum in the dosage form is at least 0.175 mg.

67. The dosage form according to any one of claims 62 to 66, wherein the amount of aluminum in the dosage form is at least 0.125 mg.

68. The dosage body according to any one of claims 62 to 67, wherein the volume of the dosage body is approximately 0.5 mL.

69. A syringe containing a drug composition according to any one of claims 1, 2, 4 to 61, or a drug according to any one of claims 62 to 68.

70. A vial containing one or more pharmaceutical compositions according to any one of claims 1, 2, 4 to 61, or administrations according to any one of claims 62 to 68.

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

72. A method for immunizing a target, the method comprising the step of administering to the target an immunologically effective amount of a pharmaceutical composition according to any one of claims 1, 2, 4 to 61, or a dosage substance according to any one of claims 62 to 68.

73. The method according to claim 71 or 72, wherein, upon administration, the pharmaceutical composition or the administered substance 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 according to claim 73, wherein the immune response is or includes an antibody and / or B cell response.

75. The method according to claim 73 or claim 74, wherein the immune response is at a level higher than a corresponding reference level, where the reference level is the level of immune response induced in a subject after administration of a reference composition.

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

77. The method according to claim 75 or claim 76, wherein the reference composition comprises the immunogenic composition and an aluminum-based adjuvant or an adjuvant containing the same, and the aluminum concentration in the reference composition is 1.25 mg / mL or more.

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

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