WO2023192997A2

WO2023192997A2 - Immunogenic compositions for b-cell recall response to a polysaccharide antigen

Info

Publication number: WO2023192997A2
Application number: PCT/US2023/065222
Authority: WO
Inventors: Richard Malley; Fan Zhang; Yingjie Lu
Original assignee: The Children's Medical Center Corporation
Priority date: 2022-03-31
Filing date: 2023-03-31
Publication date: 2023-10-05
Also published as: WO2023192997A3

Abstract

Immunogenic compositions and methods of use are described. The technology relates to methods, compositions and vaccines based on an improved MAPS multi-pathogen vaccine to mediate a T-cell independent (TI) to T-cell dependent (TD) switch of anti-CPS responses, and use of vaccine compositions comprising multiple (at least two, or 3, or 4 or more) MAPS immunogenic complexes comprising CPS polysaccharides from different pathogens for robust TD responses to more than one pathogen CPS. The multi pathogen MAPS vaccines and immunogenic compositions described herein can potentiate/mediate B cell recall responses to CPS in subjects, can induce B cell responses, and/or can increase uptake, processing, and/or presentation of a polysaccharide antigen (e.g., from a pathogen) by an antigen-presenting cell (APC).

Description

IMMUNOGENIC COMPOSITIONS FOR B-CELL RECALL RESPONSE TO A POLYSACCHARIDE ANTIGEN

CROSS-REFERENCED TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63,325,844 filed March 31, 2022, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on March 31, 2023, is named “701039-191750WOPT_SL.xml” and is 26,483 bytes in size.

BACKGROUND

[0003] Capsular polysaccharides (CPS) are important antigenic targets against bacterial infections. However, CPS can elicit short-lived immune responses in adults and are poorly immunogenic in young children. There remains a need for immunogenic compositions useful for treatment of, or protection from, bacterial infections.

SUMMARY

[0004] In general, bacterial CPS are type-II T-independent (TI) antigens - in that they activate B cells without engaging cognate T-helper (Th) cells, leading to poor antibody production and no long-lived immune memory. As a consequence, immunization with vaccines comprised of purified CPS usually induces little to no anti-CPS responses in infants or young children, and only transient antibody production in adults even when given at a high dose (e.g., 25 pg or more per CPS).

[0005] Herein, the inventors have improved upon a previous multi-component approach, referred to as MAPS (for Multiple Antigen Presenting System), where CPS are biotinylated and tightly coupled (Kd ~ 10-15 M) to pathogen-specific proteins to which an avidin-like protein (rhizavidin, rhavi) is genetically fused (Helppolainen et al., 2007, Biochem J 405:397-405; Zhang et al., 2013, Proc Natl Acad Sci U S A 110: 13564-9). MAPS, which is described in W02012/155007 and W02020/056202, which are both incorporated herein in their entirety by reference, which can induce the same T-cell dependent (TD) anti-CPS responses at a comparable, and at times superior, magnitude to those obtained with CPS-protein conjugates (Zhang et al., 2013, Proc Natl Acad Sci U S A 110: 13564-9; Anonymous, 2014, Pneumonia 3:92-121).

[0006] Herein, the inventors have improved upon MAPS for multi-pathogen vaccine development and the demonstrate the use of MAPS to mediate a T-cell independent (TI) to T-cell dependent (TD) switch of anti-CPS responses, and use of vaccine compositions comprising multiple (at least two, or 3, or 4 or more) MAPS immunogenic complexes comprising CPS polysaccharides from different pathogens for robust TD responses to more than one pathogen CPS. In some embodiments, immunogenic compositions described herein can potentiate/mediate B cell recall responses to CPS in subjects, can induce B cell responses, and/or can increase uptake, processing, and/or presentation of a polysaccharide antigen (e.g., from a pathogen) by an antigen-presenting cell (APC).

[0007] In at least one aspect, the present disclosure provides a method of potentiating a B cell recall response to a polysaccharide antigen of a pathogen to a predetermined target level, the method including: administering to a subject an immunogenic composition including: (a) a polysaccharide antigen of the pathogen; and (b) at least one polypeptide antigen that is expressed by the pathogen; where the polysaccharide antigen is associated with the at least one polypeptide antigen; and where following administration of the immunogenic composition to the subject and subsequent exposure of the subject to the pathogen, the immunogenic composition potentiates a B cell recall response to the polysaccharide antigen to the predetermined target level.

[0008] In some embodiments, the immunogenic composition includes an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and the polysaccharide antigen of the pathogen; and (b) a fusion protein including: (i) a biotin-binding moiety; and (ii) the at least one polypeptide antigen that is expressed by the pathogen, where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein. In some embodiments, the immunogenic composition includes an immunogenic conjugate, where the immunogenic conjugate includes the polysaccharide antigen of the pathogen covalently conjugated to the at least one polypeptide antigen that is expressed by the pathogen.

[0009] In some embodiments, the predetermined target level is a level that is higher than the corresponding control level. In some embodiments, the control level is a level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not include a polypeptide antigen expressed by the pathogen. In some embodiments, the predetermined target level is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not include a polypeptide antigen expressed by the pathogen. In some embodiments, the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or by killing of the pathogen by immune sera from the subject in an opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not include a polypeptide antigen expressed by the pathogen. In some embodiments, the predetermined target level is determined based on a corresponding level of a B cell recall response induced in a non-human mammalian model upon administration of the immunogenic composition to the non-human mammalian model and subsequent exposure of the non-human mammalian model to the pathogen.

[0010] In some embodiments, the method further includes measuring the level of the B cell recall response in the subject following subsequent exposure to the pathogen. In some embodiments, the measured level of the B cell recall response is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not include a polypeptide antigen expressed by the pathogen. In some embodiments, the method further includes confirming that the level of the B cell recall response after the subject has been exposed to the pathogen reaches the pre -determined target level, e.g., a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not include a polypeptide antigen expressed by the pathogen.

[0011] In some embodiments, the B cell recall response includes activation and/or generation of memory B cells that are specific for the polysaccharide antigen. In some embodiments, the B cell recall response includes activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells. In some embodiments, the B cell recall response includes activation of polysaccharide antigen-specific B cells via interaction with polypeptide antigenspecific Th cells. In some embodiments, the B cell recall response includes activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells and polypeptide antigen-specific Th cells.

[0012] In at least one aspect, the present disclosure provides a method of producing a B cell immune response to a polysaccharide antigen of a pathogen at a predetermined target level, the method including administering to a subject an immunogenic composition including an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen of the pathogen; and (b) a polypeptide including a biotin-binding moiety; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the polypeptide; and where the immunogenic complex, upon administration of the immunogenic composition to the subject, produces in the subject a B cell immune response to the polysaccharide antigen at a predetermined target level. In some embodiments, the predetermined target level is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen. In some embodiments, the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at a level that is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen. In some embodiments, the polypeptide including the biotin-binding moiety is a fusion protein including: (i) the biotin-binding moiety; and (ii) at least one polypeptide antigen. In some embodiments, the B cell immune response is or includes a MHC class II -dependent response.

[0013] In at least one aspect, the present disclosure provides a method of increasing uptake, processing, and/or presentation of a polysaccharide antigen from a pathogen by an antigen-presenting cell (APC) to a predetermined target level, the method including contacting an APC with an immunogenic composition including: (a) a polysaccharide antigen of the pathogen; and (b) a polypeptide; where the polysaccharide antigen is associated with the polypeptide; and where the immunogenic composition, upon contacting the APC, increases uptake, processing, and/or presentation of the polysaccharide antigen to the predetermined target level.

[0014] In some embodiments, the immunogenic composition includes an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and the polysaccharide antigen of the pathogen; and (b) the polypeptide, where the polypeptide includes a biotin-binding moiety; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the polypeptide. In some embodiments, the immunogenic composition includes an immunogenic conjugate, where the immunogenic conjugate includes the polysaccharide antigen of the pathogen covalently conjugated to the polypeptide. In some embodiments, the contacting includes administering to a subject the immunogenic composition.

[0015] In some embodiments, the predetermined target level is determined based on a corresponding level of uptake, processing and/or presentation of the polysaccharide antigen upon contacting an APC in vitro with the immunogenic composition. In some embodiments, the predetermined target level is at least 20% higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the predetermined target level is characterized by a level of intracellular polysaccharide antigen present in the APC being at least 5 -fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the predetermined target level is characterized by a level of polysaccharide antigen associated with the surface of the APC being at least 10-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with an antigenic polypeptide. In some embodiments, the uptake, processing, and/or presentation of the polysaccharide antigen by the APC is or includes a MHC class Il-dependent process.

[0016] In at least one aspect, the present disclosure provides, a method of selecting an immunogenic composition candidate that induces immune responses to a polysaccharide antigen to a predetermined target level, the method including: contacting an antigen-presenting cell (APC) including MHC class II molecules with an immunogenic composition candidate, where the immunogenic composition candidate includes: (a) a polysaccharide antigen; and (b) a polypeptide; where the polysaccharide antigen is associated with the polypeptide; characterizing uptake, processing, and/or presentation of the polysaccharide antigen on the MHC class II molecules by the APC, and selecting the immunogenic composition candidate as an agent useful for inducing immune responses to a polysaccharide antigen if the APC uptakes, processes, and/or presents the polysaccharide antigen on MHC class II molecules at a predetermined target level.

[0017] In some embodiments, the immunogenic composition includes an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and the polysaccharide antigen; and (b) the polypeptide, where the polypeptide includes a biotin-binding moiety; where the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the polypeptide. In some embodiments, the polypeptide including the biotin-binding moiety is a fusion protein including: (i) the biotin-binding moiety; and (ii) at least one polypeptide antigen. In some embodiments, the immunogenic composition includes an immunogenic conjugate, where the immunogenic conjugate includes the polysaccharide antigen covalently conjugated to the polypeptide.

[0018] In some embodiments, the predetermined target level is at least 2-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the predetermined target level is at least 5-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the predetermined target level is a level that is at least 10- fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the characterizing includes measuring a level of intracellular polysaccharide antigen present in the APC. In some embodiments, the characterizing includes measuring a level of polysaccharide antigen associated with the surface of the APC.

[0019] In at least one aspect, the present disclosure provides a method of immunizing a subject against a pathogen, the method including administering to a subject a dose of an immunogenic composition including an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein including: (i) a biotin-binding moiety; and (ii) at least one polypeptide antigen that is expressed by the pathogen; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; and where the administered dose is lower than that of a reference composition to achieve in the subject, upon exposure to the pathogen, an equivalent or greater B cell recall response to the polysaccharide antigen. In some embodiments, the B cell recall response of the immunized subject is characterized in that exposure of the immunized subject to the pathogen produces an antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at an equivalent or greater level to that produced by the reference composition.

[0020] In at least one aspect, the present disclosure provides a method of immunizing a subject against a pathogen, the method including administering to a subject a dose of an immunogenic composition including an immunogenic complex, where the immunogenic complex includes: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein including: (i) a biotin-binding moiety; and (ii) at least one polypeptide antigen that is expressed by the pathogen; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; and where the administered dose provides protection against the pathogen for a longer period of time than provided by the same dose of a reference composition. In some embodiments, the reference composition includes a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the reference composition does not include a polypeptide antigen expressed by the pathogen. In some embodiments, the reference composition includes a polysaccharide antigen covalently conjugated to a carrier polypeptide.

[0021] In some embodiments, the protection against the pathogen includes a B cell recall response. In some embodiments, the B cell recall response includes an antibody response against the polysaccharide antigen induced by exposure of the subject to the pathogen, and where the immunogenic composition potentiates the B cell recall response to a level at least 20% higher than the corresponding level produced by administration to the subject of the equivalent dose of an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the protection against the pathogen includes a Thl response. In some embodiments, the Thl response includes production of IFN-y and/or TNF-a by CD4+ T cells at a level that is at least 1.1-fold higher than a corresponding level of IFN-y and/or TNF-a produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the protection against the pathogen includes a Thl7 response. In some embodiments, the Thl7 response includes production of IL-17, IL- 21, IL-22, IL24 and/or IL-26 by CD4+ T cells at a level that is at least 1.1 -fold higher than a corresponding level of IL- 17, IL-21, IL-22, IL24 and/or IL-26 produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the protection against the pathogen includes a CD8 response. In some embodiments, the CD8 response includes production of IFN-y, granzyme B, and/or perforin by CD8 T cells at a level that is at least 1.1- fold higher than a corresponding level of IFN-y, granzyme B, and/or perforin produced by CD8+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition including a polysaccharide antigen that is not associated with a polypeptide antigen.

[0022] In at least one aspect, the present disclosure provides a method including: administering to a subject, who has received a prime MAPS vaccine against a pathogen, a booster vaccine including a polysaccharide antigen of the pathogen, where the MAPS vaccine includes: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein including: (i) a biotin-binding moiety; and (ii) at least one polypeptide antigen that is expressed by the pathogen; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; and where the booster vaccine, upon administration to the subject that has received the prime MAPS vaccine, induces a B cell response to the polysaccharide antigen at a predetermined target level.

[0023] In some embodiments, the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine. In some embodiments, the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine. In some embodiments, the booster vaccine including the polysaccharide antigen is or includes the MAPS vaccine. In some embodiments, the booster vaccine including the polysaccharide antigen is or includes a preparation including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the booster vaccine including the polysaccharide antigen is or includes a polysaccharide antigen covalently conjugated to a carrier polypeptide. In some embodiments, the method includes administering the MAPS vaccine to the subject prior to administering the booster vaccine.

[0024] In at least one aspect, the present disclosure provides a method including: administering to a subject, who has received a prime vaccine against a pathogen, a booster MAPS vaccine, where the MAPS vaccine includes: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein including: (i) a biotin-binding moiety; and (ii) at least one polypeptide antigen that is expressed by the pathogen; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein; where the prime vaccine includes a polysaccharide antigen of the pathogen; and where the booster MAPS vaccine, upon administration to the subject who has received the prime vaccine, induces a B cell response to the polysaccharide antigen at a predetermined target level.

[0025] In some embodiments, the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein. In some embodiments, the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine including a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein. In some embodiments, the prime vaccine including the polysaccharide antigen is or includes the MAPS vaccine. In some embodiments, the prime vaccine including the polysaccharide antigen is or includes a preparation including a polysaccharide antigen that is not associated with a polypeptide antigen. In some embodiments, the prime vaccine including the polysaccharide antigen is or includes a polysaccharide antigen of covalently conjugated to a carrier polypeptide. In some embodiments, the method includes administering the prime vaccine to the subject prior to administering the booster MAPS vaccine. In some embodiments, the B cell response is a T helper (Th)-dependent response against the polysaccharide antigen and/or the polypeptide antigen.

[0026] In some embodiments, the pathogen is a Streptococcal (e.g., Group A, Group B, and Viridans), Staphylococcal (e.g., S. aureus), Meningococcal, Pneumococcal, Gram-Negative Bacteria (e.g., E. coli, Klebsiella, Pseudomonas, Enterobacter, Citrobacter, Acinetobacter, Serratia, Burkholderia, Salmonella, Shigella, and Bordetella), coronavirus, Mycobacterium (e.g., M. tuberculosis), Plasmodium (e.g., P. falciparum), pathogen. In some embodiments, the immunogenic composition includes a plurality of different species of immunogenic complexes, where the different species include different polysaccharide antigens, and/or different polypeptide antigens. In some embodiments, the polysaccharide antigen is or includes a portion of a capsular polysaccharide of Streptococcus pneumoniae. In some embodiments, the capsular polysaccharide of Streptococcus pneumoniae is selected from: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 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 polypeptide antigen is a polypeptide antigen selected from pneumococcal antigens (e.g., Group A, Group B, and Viridans antigens), tuberculosis antigens, anthrax antigens, HIV antigens, seasonal or epidemic flu antigens, Pertussis antigens, Staphylococcus aureus antigens, Meningococcal antigens, Haemophilus antigens, HPV antigens, Shigella antigens, Salmonella antigens, malaria antigens, Pseudomonas antigens, coronavirus antigens, or combinations thereof. In some embodiments, the polypeptide antigen is an SP1500 polypeptide, an SP0785 polypeptide, and/or a pneumolysin polypeptide.

[0027] In some embodiments, the immunogenic composition is administered as part of a pharmaceutical composition further including a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further includes one or more adjuvants. In some embodiments, the one or more adjuvants are or include a co-stimulation factor. In some embodiments, the one or more adjuvants are selected from the group consisting of aluminum phosphate, aluminum hydroxide, and phosphated aluminum hydroxide. In some embodiments, the one or more adjuvants are or include aluminum phosphate. In some embodiments, the pharmaceutical composition is formulated for injection.

[0028] In some embodiments, the biotin-binding moiety is or includes a dimeric biotin-binding moiety. In some embodiments, the dimeric biotin-binding moiety is or includes a rhizavidin polypeptide. In some embodiments, the rhizavidin polypeptide includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or a biotin-binding fragment thereof.

[0029] In at least one aspect, the present disclosure provides an immunogenic complex including: (a) a biotinylated polysaccharide antigen including biotin and a polysaccharide antigen; and (b) a biotinbinding polypeptide; where the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding polypeptide; and where the biotin-binding polypeptide does not include a polypeptide antigen from a pathogen. However, in some embodiments, the biotin-binding polypeptide can include a polypeptide antigen from a tumor or tissue. In some embodiments, the biotin-binding polypeptide is or includes avidin. In some embodiments, the biotin-binding polypeptide is or includes rhizavidin.

[0030] Other aspects relate to vaccine compositions comprising at least two, or at least three or at least 4, or at least 5 immunogenic compexes as described herein. Other aspects relate to fusion proteins, including, but not limited to fusion protein comrprising a biotin-binding polypeptide comprising rhizavidin and a polypeptide antigen comprising PdT. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0033] Figure 1A shows relative IgM and IgG antibody levels against CPS 14 for mice immunized with adjuvant alone (Alum), or adjuvanted CPS 14 (type 14 pneumococcal CPS), CPS 14 MAPS (MAPS), or CPS 14 conjugate vaccine (CV). Antibody levels were measured after one (Pl), two (P2), and three (P3) immunizations. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann- Whitney U test are also shown on the graph.

[0034] Figure IB shows the avidity of IgG antibodies against CPS 14 for mice immunized with adjuvanted CPS 14 MAPS (MAPS) or CPS 14 conjugate vaccine (CV) after two (P2) or three (P3) immunizations.

[0035] Figure 1C shows relative IgM and IgG antibody levels against CPS 14 for wild type (WT) and MHCIT" mice immunized with CPS 14 MAPS after one (Pl) or two (P2) immunizations. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0036] Figure ID shows relative IgM and IgG antibody levels against CPS 14 for Ragl-/- mice that received an adoptive transfer with splenocytes isolated from naive (SpN) or CPS 14 MAPS immunized mice (SpM). IgG levels were measured before (Pre) and after (Post) subsequent immunization with CPS 14 or CPS 14 MAPS (MAPS). Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0037] Figure 2A shows intracellular and surface-associated CPS content for peritoneal macrophages, isolated from mice, that were incubated at 4 °C for 2 hours in a culture medium containing no CPS (Con), CPS14, or CPS14 MAPS. CPS content was measured by inhibition ELISA and normalized to the total cellular protein content. Mean and SEM are shown on the graph. [0038] Figure 2B shows intracellular and surface-associated CPS content for peritoneal macrophages, isolated from mice, that were incubated at 37 °C for 0.5, 6, or 18 hours in a culture medium containing CPS14 or CPS14 MAPS. CPS content was measured by inhibition ELISA and normalized to the total cellular protein content. Mean and SEM are shown on the graph.

[0039] Figure 2C shows intracellular and surface-associated CPS content for peritoneal macrophages isolated, from wild type (WT) or MHCIT - mice, that were incubated at 37 °C for 18 hours in a culture medium containing no CPS (Con), CPS14, heat-killed type 14 pneumococci (Pnl4), or CPS14 MAPS. CPS content was measured by inhibition ELISA and normalized to the total cellular protein content. Mean and SEM are shown on the graph. Results of statistical analyses performed using the Mann- Whitney U test are also shown on the graph.

[0040] Figure 2D shows results of a co-immunoprecipitation using cell lysates prepared from peritoneal macrophages that were incubated at 37 °C for 18 hours in a culture medium containing CPS 14 MAPS (MAPS), CPS 14 (CPS), or avidin (Avi). Co-immunoprecipitation from the cell lysates was performed using rabbit anti-CPS14 serum. The input cell lysate and resulting co-immunoprecipitated material was subject to Western blot using b-actin antibody (as an internal control) and MHC-II antibody.

[0041] Figure 3 shows relative IgG levels against CPS1, CPS3, CPS4, CPS5, and CPS14 for Ragl ^/_ mice that received an adoptive transfer with B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), CPI-primed mice (TC), or 5V-MAPS1- primed mice (TM), and were subsequently immunized with 5V-MAPS1. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0042] Figure 4 shows relative IgG levels against CPS1, CPS3, CPS4, CPS5, and CPS14 for Ragl ^/_ mice that received an adoptive transfer with B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), CPI-primed mice (TC), or 5V-MAPS1- primed mice (TM), and were subsequently immunized with 5V-MAPS2. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0043] Figure 5 shows relative IgG levels against CPS1, CPS3, CPS4, CPS5, and CPS14 for RagT^/_ mice that received an adoptive transfer with B and CD4+ T cells isolated from naive mice (BN+TN); or with B cells isolated from 5V-MAPS1 -primed mice (BM), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), CPI -primed mice (TC), or 5 V-MAPS1 -primed mice (TM); and were subsequently immunized with 5V-MAPS 1. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0044] Figure 6A shows relative IgG levels against pneumolysin (Ply) for mice immunized with adjuvant (Alum) alone (groups 1 and 2) or with adjuvanted CPS4 MAPS vaccine (groups 3 and 4). IgG levels were measured before (Pre-SP exposure) and after (Post-SP exposure) subsequent exposure to heat-killed wild type (WT) groups 1 and 3) or pneumolysin knockout (APly) (groups 2 and 4) Tigr4 strain via intraperitoneal injection. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0045] Figure 6B shows relative IgG levels against CPS4 for mice immunized with adjuvant (Alum) alone (groups 1 and 2) or with adjuvanted CPS4 MAPS vaccine (groups 3 and 4). IgG levels were measured before (Pre-SP exposure) and after (Post-SP exposure) subsequent exposure to heat-killed wild type (WT) groups 1 and 3) or pneumolysin knockout (APly) (groups 2 and 4) Tigr4 strain via intraperitoneal injection. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann- Whitney U test are also shown on the graph.

[0046] Figure 7 shows relative IgG levels against fusion protein 1 (CPI) for naive mice and mice immunized with CPI or 5V-MAPS1. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann-Whitney U test are also shown on the graph.

[0047] Figure 8 shows IL-5 levels measured using ELISA in supernatants of peripheral blood samples from naive mice or mice immunized with rhizavidin (Rhavi) after ex vivo stimulation with Rhavi or egg avidin (Egg avi).

[0048] Figure 9 shows relative IgG levels against fusion protein 1 (CPI) and avidin (Avi) in Ragl ^/_ mice that received an adoptive transfer with B cells purified from naive mice (BN), alone or in combination with CD4⁺ T cells purified from naive mice (TN), or CPI-primed mice (TC), or 5V- MAPS1 -primed mice (TM), and were subsequently immunized with 5V-MAPS2. Relative geometric mean antibody titer and 95% confidence intervals are shown on the graph as arbitrary units (a.u.). Results of statistical analyses performed using the Mann- Whitney U test are also shown on the graph.

[0049] Figure 10 shows a Western blot of heat-killed wild-type or pneumolysin knockout (APly) Tigr4 pneumococci. Pneumolysin, and SP0785 and SP1500 (as controls), was detected using rabbit immune sera. [0050] Figure 11 shows the results of in vitro biochemical analysis of the stability of an exemplary MAPS complex. A MAPS complex including biotinylated CPS 14 and avidin (MAPS) was compared to avidin alone. Samples were subjected to boiling and/or proteinase K (ProK) treatment. Treated samples were analyzed on a gel. Labeled arrows indicate expected positions for ProK, avidin not present in a MAPS complex (Avi), and partially digested avidin (Avi F). Intact MAPS complex was observed in lanes 1 and 3 (see unlabeled arrows).

[0051] Figure 12 is a schematic representation of an exemplary Multiple Antigen Presenting System (MAPS). In the exemplary embodiment shown, MAPS immunogenic complexes comprise one or more polypeptide antigens fused to the biotin-binding protein rhizavidin, or a biotin-binding domain or biotinbinding fragment thereof, and a biotinylated antigenic polysaccharide. In this figure, each MAPS complex is formed between one or more fusion proteins and a biotinylated polysaccharide by non- co valent binding of a truncated rhizavidin to biotin.

[0052] Figure 13 shows exemplary structures and chemical information for exemplary polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, and 14.

CERTAIN DEFINITIONS

[0053] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0054] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0055] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastrical, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0056] Amino acid: In its broadest sense, the term “amino acid”, as used herein, refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N- C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D- amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0057] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kDa tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kDa each) and two identical light chain polypeptides (about 25 kDa each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g. , attachment of a glycan, a pay load [e.g. , a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]).

[0058] Antigen: The term “antigen”, as used herein, refers to (i) an agent that induces an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen induces a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen induces a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen induces a humoral response and a cellular response. In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide (herein also referred to as a “polypeptide antigen”), a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer (e.g. , other than a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a polysaccharide. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g. , together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen. In some embodiments, an antigen is a polypeptide or a polysaccharide that, upon administration to a subject, induces a specific and/or clinically relevant immune response to such polypeptide or polysaccharide. In some embodiments, an antigen is selected to induce a specific and/or clinically relevant immune response to such polypeptide or polysaccharide. [0059] Associated with: Two entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0060] Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[0061] Carrier protein: As used herein, the term “carrier protein” refers to a protein or peptide that is coupled, or complexed, or otherwise associated with a hapten (e.g., a small peptide or lipid) or immunogenic antigen (e.g., a polysaccharide) and that induces or improves an immune response to such a coupled, or complexed, or otherwise associated hapten (e.g., a small peptide or lipid) or immunogenic antigen (e.g. , a polysaccharide). In some embodiments, a carrier protein is not itself the intended target for immune response. In some embodiments, a carrier protein can induce an immune response. In some embodiments, such an immune response is or comprises a response to a hapten or immunogenic polysaccharide antigen that is coupled, or complexed, or otherwise associated with such a carrier protein. In some embodiments, such an immune response is or comprises a response to both a carrier protein and a hapten or immunogenic polysaccharide antigen that is coupled, or complexed, or otherwise associated with such a carrier protein. In some embodiments, no significant immune response to a carrier protein itself occurs. In some embodiments, immune response to a carrier protein may be detected; in some embodiments, immune response to such a carrier protein is strong. In some embodiments, a carrier protein is coupled, or complexed, or otherwise associated with one or more other molecules. An exemplary carrier protein is the diphtheria CRM197 carrier protein, e.g., of PCV20. In some embodiments, the term “carrier protein” does not refer to a polypeptide antigen as that term is disclosed herein, e.g., a polypeptide antigen that is from the same pathogen as a polysaccharide antigen and associated with the polysaccharide. [0062] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is exposed to two or more therapeutic regimens (e.g. , two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0063] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e. , with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0064] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0065] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment includes a discrete portion of the whole which discrete portion shares one or more functional characteristics found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment of a polymer, e.g., a polypeptide or a polysaccharide, comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0066] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%,

50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0067] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0068] Improve, increase, inhibit or reduce: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (e.g. , prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0069] Immunologically effective amount or immunologically effective dose: As used herein, “immunologically effective amount” or “immunologically effective dose” refers to an amount of an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, which when administered to a subject, either in a single dose or as part of a series of doses, that is sufficient to enhance a subject’s own immune response against a subsequent exposure to a pathogen. An immunologically effective amount may vary based on the subject to be treated, the species of the subject, the degree of immune response desired to induce, etc. In some embodiments, an immunologically effective amount is sufficient for treatment or protection of a subject having or at risk of having disease. In some embodiments, an immunologically effective amount refers to a non-toxic but sufficient amount that can be an amount to treat, attenuate, or prevent infection and/or disease (e.g., a sign or symptom associated with infection and/or disease) in any subject. In some embodiments, an immunologically effective amount is sufficient to induce an immunoprotective response upon administration to a subject.

[0070] Immunoprotective response or protective response: As used herein, “immunoprotective response” or “protective response” refers to an immune response that mediates antigen or immunogen- induced immunological memory. In some embodiments, an immunoprotective response is induced by the administration of a substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition to a subject. In some embodiments, immunoprotection involves one or more of active immune surveillance, a more rapid and effective response upon immune activation as compared to a response observed in a naive subject, efficient clearance of the activating agent or pathogen, followed by rapid resolution of inflammation. In some embodiments, an immunoprotective response is an adaptive immune response. In some embodiments, an immunoprotective response is sufficient to protect an immunized subject from productive infection by a particular pathogen or pathogens to which a vaccine is directed (e.g. , SARS-CoV-2 nfection).

[0071] Immunization: As used herein, “immunization”, or grammatical equivalents thereof, refers to a process of inducing an immune response to an infectious organism or agent in a subject (“active immunization”), or alternatively, providing immune system components against an infectious organism or agent to a subject (“passive immunization”). In some embodiments, immunization involves the administration of one or more antigens, immunogens, immunogenic complexes, vaccines, immune molecules such as antibodies, immune sera, immune cells such as T cells or B cells, or pharmaceutical compositions to a subject. In some embodiments, immunization is performed by administering an immunologically effective amount of a substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, immune molecule such as an antibody, immune serum, immune cell such as a T cell or B cell, or pharmaceutical composition to a subject. In some embodiments, immunization results in an immunoprotective response in the subject. In some embodiments, active immunization is performed by administering to a subject an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, vaccine, or pharmaceutical composition. In some embodiments, passive immunization is performed by administering to a subject an immune system component, e.g., an immune molecule such as an antibody, immune serum, or immune cell such as a T cell or B cell.

[0072] Isolated: As used herein, the term “isolated”, or grammatical equivalents thereof, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polysaccharide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide or polysaccharide. Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide or polysaccharide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.

[0073] Linker: As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form Sl- L-S2, wherein S 1 and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (Holliger et al, 1993; Poljak, 1994). [0074] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0075] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0076] Polysaccharide: The term “polysaccharide” as used herein refers to a polymeric carbohydrate molecule composed of long chains of monosaccharide units bound together by glycosidic, phosphodiester, or other linkages and on hydrolysis give the constituent monosaccharides or oligosaccharides. Polysaccharides range in structure from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, structural polysaccharides such as cellulose and chitin and microbial polysaccharides, and antigenic polysaccharides found in microorganisms including, but not limited to, capsular polysaccharides (CPS), O polysaccharides (OPS), core O polysaccharides (COPS), and lipopolysaccharides (LPS).

[0077] Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids, e.g, linked to each other by peptide bonds. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0078] Prevention: The term “prevent” or “prevention”, as used herein in connection with a disease, disorder, and/or medical condition, refers to reducing the risk of developing the disease, disorder and/or condition, and/or a delay of onset, and/or reduction in frequency and/or severity of one or more characteristics or symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a pre-defined period of time.

[0079] Protein: As used herein, the term “protein” encompasses a polypeptide. Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain l-amino acids, d-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0080] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0081] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, subject, population, sample, sequence or value of interest is compared with a reference or control agent, animal, subject, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0082] Response: As used herein, a “response” to treatment may refer to any beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment. Such alteration may include stabilization of the condition (e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc. Subject response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of biomarkers in a sample obtained from a subject, cytology, and/or histology. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of subjects and/or tumors, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria. [0083] Risk: As will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular subject will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from subjects comparable to a particular subject. In some embodiments, relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8,

9, 10, or more.

[0084] Serotype: As used herein, the term “serotype”, also referred to as a serovar, refers to a distinct variation within a species of bacteria or virus or among immune cells of different subjects. These microorganisms, viruses, or cells are classified together based on their cell surface antigens, allowing the epidemiologic classification of organisms to the sub-species level. A group of serovars with common antigens may be referred to as a serogroup or sometimes serocomplex.

[0085] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is a subject to whom diagnosis and/or therapy is and/or has been administered.

[0086] Susceptible to: A subject who is “susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition is a subject who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of subjects suffering from the disease, disorder, or condition).

[0087] Symptoms are reduced: As used herein, “symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency, e.g. , to a statistically and/or clinically significant or relevant level. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

[0088] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0089] Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent. For the purposes of the present invention, vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition. In some embodiments, vaccination initiates immunization. In some embodiments, vaccination elicits protection from infection with a disease-causing agent. In some embodiments, vaccination provides treatment of an infection with a disease-causing agent.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0090] The present disclosure relates, generally, to compositions, systems, and methods that include complexed proteins and polysaccharides, e.g., vaccines of complexed proteins and polysaccharides.

Such complexes can be used, e.g., to induce and/or increase an immunoprotective response in subjects at risk of or suffering from pathogenic infection.

Overview

[0091] Capsular polysaccharides (CPS) are the main constituent of bacterial capsules and play important roles in maintaining bacterial structure, facilitating the adherence to host cells, and preventing the complement-mediated opsonic killing by phagocytes (Roberts, 1996, Annu Rev Microbiol 50:285- 315; Moradali et al., 2020, Nat Rev Microbiol 18: 195-210; Wen, 2015, Chapter 3 - Bacterial Capsules, p 33-53. In Tang Y-W, Sussman M, Liu D, Poxton I, Schwartzman J (ed), Molecular Medical Microbiology (Second Edition) doi:https://doi.org/10.1016/B978-0-12-397169-2.00003-2). Generating functional antibodies to CPS can protect against mucosal acquisition and invasive disease due to encapsulated bacterial pathogens (Malley et al., 1998, J Infect Dis 178:878-82; Mitsi et al., 2017, Mucosal Immunology 10:385-394; Sadarangani, 2018, Front Immunol 9:2674). Therefore, CPS have been used as important antigen targets for many bacterial vaccines. However, with few exceptions (Kalka-Moll et al., 2002, J Immunol 169:6149-53; Sun et al., 2016, Glycobiology 26: 1029-1040), most bacterial CPS are type-II T-independent (TI) antigens: they activate B cells (by cross-linking surface receptors) without engaging cognate T-helper (Th) cells, leading to poor antibody production and no long-lived immune memory. As a consequence, immunization with vaccines comprised of purified CPS usually induces little to no anti-CPS responses in infants or young children, and only transient antibody production in adults even when given at a high dose (e.g., 25 pg or more per CPS).

[0092] The development of polysaccharide-protein conjugate vaccines, comprising CPS covalently linked to protein carriers (Avery et al., 1929, J Exp Med 50:533-50), has overcome this problem. Conjugate vaccines induce robust anti-CPS responses in infants and provide effective protection against invasive disease caused by encapsulated bacterial pathogens, including Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis types A, C, W135 and Y, and most recently, Salmonella typhimurium (Alicino et al., 2017, Vaccine 35:5776-5785; Heath, 1998, Pediatr Infect Dis J 17: S 117-22; Harrison et al., 2010, Nat Rev Drug Discov 9:429-30; Yousafzai et al., 2021, Lancet Glob Health 9:e 1154-e 1162). Studies revealed immunological properties of conjugate-induced anti-CPS responses, including Ig class switching (from IgM to IgG), Ig affinity-maturation, MHCII-dependency, and immune memory generation (Sun et al., 2016, Glycobiology 26: 1029-1040; Rose et al., 2005, Clin Diagn Lab Immunol 12: 1216-22; Avci et al., 2011, Nat Med 17: 1602-9; Colino et al., 2013, J Immunol 191:3254-63; Rappuoli, 2018, Sci Transl Med 10), indicating that CPS-protein conjugates, in contrast to pure CPS, can activate CPS-specific B cells (BCPS) via a classical T-dependent (TD) pathway.

[0093] Following the success of polysaccharide-protein conjugates, other CPS/protein vaccines using different types of association between CPS and protein carriers have been proposed and developed. One platform uses a protein matrix onto which the CPS is non-specifically adsorbed: in preclinical studies, such a construct was shown to induce IgG antibodies, a feature of TD responses, to two studied CPS antigens (Thanawastien et al., 2015, Proceedings of the National Academy of Sciences 112:E1143- E1151). In another approach called MAPS (for Multiple Antigen Presenting System), CPS are biotinylated and tightly coupled (Kd ~ 10-15 M) to pathogen-specific proteins to which an avidin-like protein (rhizavidin, rhavi) is genetically fused (Helppolainen et al., 2007, Biochem J 405:397-405; Zhang et al., 2013, Proc Natl Acad Sci U S A 110: 13564-9). MAPS can induce the same TD anti-CPS responses at a comparable, and at times superior, magnitude to those obtained with CPS-protein conjugates (Zhang et al., 2013, Proc Natl Acad Sci U S A 110: 13564-9; Anonymous, 2014, Pneumonia 3:92-121). A MAPS vaccine at a dose of 1, 2, or 5 pg of each of 24 pneumococcal polysaccharides and a pneumococcal -rhavi fusion protein carrier was shown to generate robust functional anti-CPS IgG antibodies in healthy young and older adults, with comparable (and in some cases, superior) immunogenicity to the licensed 13- valent pneumococcal vaccine, Prevnar 13, for the common serotypes (Chichili et al., 2020, Open Forum Infectious Diseases 7:S640-S640).

[0094] Immunogenic compositions described herein can, in some aspects, mediate a TI to TD switch of anti-CPS responses. In some embodiments, immunogenic compositions described herein can potentiate/mediate B cell recall responses in subjects, can induce B cell responses, and/or can increase uptake, processing, and/or presentation of a polysaccharide antigen (e.g., from a pathogen) by an antigen- presenting cell (APC).

Immunogenic Complexes

[0095] In some embodiments, the present disclosure encompasses immunogenic complexes that include one or more polypeptides and one or more polysaccharides.

[0096] In some embodiments, immunogenic complexes are, or are based on, Multiple Antigen Presenting System (MAPS) complexes. Aspects of the MAPS platform have been previously described in WO2012/155007 and W02020/056202, the contents of which are herein incorporated by reference in their entirety, and are shown schematically in Figure 12. See also Zhang et al., 2013, Proc Natl Acad Sci U S A 110: 13564-9.

[0097] The technology disclosed herein is based on the discovery that when a polysaccharide antigen is attached to a polypeptide antigen (e.g., a fusion protein comprising one or more polypeptide antigens) that is expressed by the same pathogen as the polysaccharide antigen, there is a significant increase or potentiation of the recall immune response against the pathogen, as compared to when the polypeptide antigen is not from the same pathogen.

[0098] As described herein, immunogenic complexes of the disclosure include one or more polypeptides (e.g., antigenic polypeptides) non-covalently complexed with one or more polysaccharides (e.g., antigenic polysaccharides). In some embodiments, the antigenic polypeptides are expressed by the same pathogen as the antigenic polysaccharides. In some embodiments, the antigenic polypeptides are expressed by the same serotype of the same pathogen. In some embodiments, one or more antigenic polypeptides are complexed via affinity interaction with one or more antigenic polysaccharides. In some embodiments, immunogenic complexes of the disclosure include one or more antigenic polypeptides non-covalently complexed with one or more antigenic polysaccharides using one or more affinity molecule/complementary affinity molecule pairs. In some embodiments, an immunogenic complex includes (i) a first affinity molecule described herein conjugated to one or more antigenic polysaccharides, and (ii) a fusion protein that is or comprises a complementary affinity molecule described herein and at least one polypeptide. Upon association of the first affinity molecule and the complementary affinity molecule, the one or more antigenic polypeptides are non-covalently complexed to the one or more antigenic polysaccharides.

[0099] In some embodiments, one or more antigenic polypeptides are complexed via affinity interaction with one antigenic polysaccharide. In some embodiments, immunogenic complexes of the disclosure include one or more antigenic polypeptides non-covalently complexed with one antigenic polysaccharide using one affinity molecule/complementary affinity molecule pair. In some embodiments, immunogenic complexes of the disclosure include one or more antigenic polypeptides non-covalently complexed with one antigenic polysaccharide using one or more affinity molecule/complementary affinity molecule pairs. In some embodiments, each of the affinity molecule/complementary affinity molecule pairs is the same, e.g., biotin/biotin-binding moiety pairs. In some embodiments, an immunogenic complex includes (i) a first affinity molecule described herein conjugated to one antigenic polysaccharide, and (ii) a fusion protein that is or comprises a complementary affinity molecule described herein and at least one polypeptide (e.g., antigenic polypeptide described herein). Upon association of the first affinity molecule and the complementary affinity molecule, the one or more antigenic polypeptides are non-covalently complexed to the one antigenic polysaccharide.

[0100] In some embodiments, the affinity molecule/complementary affinity molecule pair is selected from one or more of biotin/biotin-binding moiety, antibody/antigen, enzyme/substrate, receptor/ligand, metal/metal-binding protein, carbohydrate/carbohydrate binding protein, lipid/lipid-binding protein, and His tag/His tag -binding molecule.

[0101] In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof), and the complementary affinity molecule is a moiety, e.g., a biotin-binding protein, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is rhizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, NeutrA vidin, CaptA vidin™, or a biotin-binding domain or biotin-binding fragment thereof, or a combination thereof. In some embodiments, the biotin-binding moiety is rhizavidin, or a biotin-binding domain or biotinbinding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 1, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety is or comprises a polypeptide of SEQ ID NO: 2, or a biotinbinding domain or biotin-binding fragment thereof. [0102] In some embodiments, the one or more antigenic polysaccharides comprise one or more affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the one or more affinity molecules comprise biotin or biotin derivatives.

[0103] In some embodiments, the antigenic polysaccharides comprise a plurality of affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the affinity molecules comprise biotin or biotin derivatives.

[0104] In some embodiments, one or more antigenic polypeptides are covalently linked (e.g., fused) to a complementary affinity molecule described herein. In some embodiments, a fusion protein comprises one or more antigenic polypeptides and a complementary affinity molecule disclosed herein. In some embodiments, the complementary affinity molecule is or comprises a biotin-binding moiety. In some embodiments, the biotin-binding moiety comprises rhizavidin or a biotin-binding portion thereof.

[0105] In some embodiments, antigenic polysaccharides and/or antigenic polypeptides that may be included in immunogenic complexes are recombinantly or synthetically produced. In some embodiments, antigenic polysaccharides and/or antigenic polypeptides that may be included in immunogenic complexes are isolated and/or derived from natural sources. In some embodiments, antigenic polysaccharides and/or antigenic polypeptides that may be included in immunogenic complexes are isolated from viruses or from bacterial cells. Exemplary polysaccharides and/or polypeptides are described below.

Antigenic Polypeptides

[0106] In some embodiments, an immunogenic complex described herein comprises one or more polypeptide antigens. In some embodiments, a polypeptide antigen is a bacterial polypeptide, a fungal polypeptide, a tumor polypeptide, and/or a viral polypeptide. In some embodiments, a polypeptide antigen is a polypeptide of, or derived from, a gram-negative bacteria or a gram-positive bacteria. In some embodiments, a polypeptide antigen is, or is derived from, a pneumococcal (e.g., group A, group B, and viridans) antigen, a tuberculosis antigen, an anthrax antigen, an HIV antigen, an influenza (e.g., seasonal or epidemic) antigen, a pertussis antigen, a staphylococcal (e.g., S. aureus) antigen, a meninigococcal antigen, a haemophilus antigen, a HPV antigen, a Shigella antigen, a Salmonella antigen, a malaria antigen, a Pseudomonas antigen, a Klebsiella antigen, an E. coli antigen, a coronavirus antigen, or combinations thereof. In some embodiments, a polypeptide antigen is a polypeptide of, or derived from, .S', pneumoniae. In some embodiments, the one or more polypeptide antigens comprise two or more polypeptide antigens from the same bacteria, fungi, tumor, or virus. In some embodiments, the one or more polypeptide antigens comprise (i) a polypeptide of, or derived from, a first bacteria, fungi, tumor, or virus, and (ii) a polypeptide of, or derived from, a second bacterial, fungi, tumor or virus. In some embodiments, an immunogenic complex includes one or more of the following .S', pneumoniae antigenic polypeptides, or portions thereof.

Pneumolysin Polypeptides

[0107] Pneumolysin (Ply) is a .S'. pneumoniae protein toxin. In some embodiments, Ply polypeptide is a cholesterol-dependent toxin of the thiol-activated cytolysin family. In some embodiments, a Ply polypeptide is or comprises a full-length Ply polypeptide. For example, in some embodiments, a full- length Ply polypeptide has 470 amino acids (53 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, a Ply polypeptide includes a portion of a Ply polypeptide (e.g., a portion of a Ply polypeptide of SEQ ID NO: 3, which portion includes at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more contiguous amino acids of SEQ ID NO: 3). In some embodiments, a portion of an Ply polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a Ply polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and/or insertion) from a naturally-occuring wild-type Ply polypeptide sequence. For example, a Ply polypeptide may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 3 or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids of the sequence shown in SEQ ID NO: 3). Alternatively, a Ply polypeptide may contain a portion (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids) of a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 3. In some embodiments, a nucleotide sequence encoding a Ply polypeptide is provided herein as SEQ ID NO: 13.

[0108] Pneumolysins are exotoxins produced by bacteria that can cause hemolytic activity and complement activation. While highly immunogenic, their use in vaccines is limited because they cause lysis of red blood cells. Accordingly, in another aspect, provided herein are variants of .S', pneumoniae pneumolysin (Ply), its fusion construct with a biotin-binding protein, and its uses. In some embodiments, such variants, designated herein as mutant Ply or “mPly” are substantially non-hemolytic. As used herein, the phrase “substantially non-hemolytic” means the ability of lysing red blood cells being reduced by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or up to 100%, as compared to that of an equivalent concentration of a reference Ply (e.g., a wild-type Ply). In some embodiments, hemolytic activity of substantially non-hemolytic Ply is at least 5%, at least 10%, at least 15%, at least 20%, at least 20%, at least 30%, at least 30%, at least 35%, least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% lower than an equivalent concentration of a reference Ply (e.g., a wildtype Ply). In some embodiments, the substantially non-hemolytic Ply has no detectable hemolytic activity. The term “wild-type Ply” is accorded the usual definition associated with such phrase, e.g., in some embodiments a naturally occurring Ply (e.g., a Ply that is naturally secreted by a capable bacterial source). In some embodiments, a wild-type Ply protein is represented by the amino acid sequence as set forth in SEQ ID NO: 3.

[0109] In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 3) or an antigenic fragment thereof, with one or more amino acid substitutions. In some embodiments, a mutant Ply (e.g. , nonhemolytic Ply) comprises a wild-type Ply amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 3) or an antigenic fragment thereof, with one or more of the following amino acid substitutions: residue D385 substituted with N; residue C428 substituted with G, and residue W433 substituted with F. See, for example, Berry et al., “Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae” . Infect Immun. 1995 63(5): 1969-1974). In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) carrying the amino acid substitutions D385N, C428G, and W433F is referred to as PdT. In some embodiments, a PdT is or comprises the amino acid sequence as set forth in SEQ ID NO: 4.

[0110] In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) is a portion of a PdT polypeptide (e.g., a portion of the PdT polypeptide of SEQ ID NO: 4, which portion includes at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more contiguous amino acids of SEQ ID NO: 4. In some embodiments, such a portion of PdT polypeptide include the four amino acid substitutions described herein. In some embodiments, a portion of a PdT polypeptide corresponds to a protein having amino acids 2-470 of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a mutant Ply (e.g., nonhemolytic Ply) contains one or more amino acid alterations (e.g., deletion, substitution, and/or insertion) from the PdT polypeptide sequence of SEQ ID NO: 4. For example, a mutant Ply (e.g., non-hemolytic Ply) may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 4 or a portion thereof (e.g., at least 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids of the sequence shown in SEQ ID NO: 4). Alternatively, a mutant Ply (e.g., non-hemolytic Ply) may contain a portion (e.g., at least 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or more consecutive amino acids) of a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 4. In some embodiments, a mutant Ply (e.g., non-hemolytic Ply) may comprises no more than 25 (including, e.g., no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2) amino acid modifications (e.g., deletion, substitution, and/or insertion) within the sequence of SEQ ID NO: 4 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N- terminal portion and/or C-terminal portion.

SP0785 Polypeptides

[oni] SP0785 is a conserved hypothetical .S', pneumoniae protein, for example, in some embodiments as described in WO 2014/124228, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an SP0785 polypeptide is an efflux transporter protein conserved across .S', pneumoniae strains. In some embodiments, an SP0785 polypeptide is or comprises a full-length SP0785 polypeptide. For example, in some embodiments, a full-length SP0785 polypeptide has 399 amino acids (38 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 9. Without wishing to be bound by a particular theory, amino acids 1-32 of SEQ ID NO: 9 are predicted to be a signal sequence and transmembrane domain of an SP0785 polypeptide (amino acids 1-32 of the full-length protein). Accordingly, in some embodiments, an SP0785 polypeptide may exclude such a signal sequence and transmembrane domain (e.g., may comprise or consist of SEQ ID NO: 10). In some embodiments, an SP0785 polypeptide includes a portion of an SP0785 polypeptide (e.g., a portion of the SP0785 polypeptide of SEQ ID NO: 9, which portion includes at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or more contiguous amino acids of SEQ ID NO:9). In some embodiments, a portion of an SP0785 polypeptide corresponds to a protein having amino acids 33-399 of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, an SP0785 polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and/or insertion) from a naturally-occurring wild-type SP0785 polypeptide sequence. For example, an SP0785 polypeptide may contain an amino acid sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 9 or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or more consecutive amino acids of the sequence shown in SEQ ID NO: 9). Alternatively, an SP0785 polypeptide may contain a portion (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids) of a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 9. In some embodiments, a nucleotide sequence encoding an SP0785 polypeptide is provided herein as SEQ ID NO: 17. In some embodiments, an SP0785 polypeptide may comprises no more than 25 (including, e.g., no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2) amino acid modifications (e.g., deletion, substitution, and/or insertion) within the sequence of SEQ ID NO: 9 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N- terminal portion and/or C-terminal portion.

SP 1500 Polypeptides

[0112] SP1500 is a .S'. pneumoniae protein, for example, in some embodiments as described in WO

2014/124228, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, an SP1500 polypeptide is an Amino Acid ABC Transporter, amino acidbinding polypeptide conserved across .S', pneumoniae strains. In some embodiments, an SP1500 polypeptide is or comprises a full-length SP1500 polypeptide. For example, in some embodiments, a full-length SP1500 polypeptide has 278 amino acids (28 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO: 11. Without wishing to be bound by a particular theory, amino acids 1-26 of SEQ ID NO: 11 are predicted to be a signal sequence of an SP1500 polypeptide (amino acids 1-26 of the full-length protein). Accordingly, in some embodiments, a SP1500 polypeptide may exclude such a signal sequence (e.g., may comprise or consist of SEQ ID NO: 12). In some embodiments, an SP1500 polypeptide includes a portion of an SP1500 polypeptide (e.g., a portion of the SP 1500 polypeptide of SEQ ID NO: 11, which portion includes at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or more contiguous amino acids of SEQ ID NO: 11). In some embodiments, a portion of an SP1500 polypeptide corresponds to a protein having amino acids 27-278 of the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, an SP 1500 polypeptide contains one or more amino acid alterations (e.g., deletion, substitution, and/or insertion) from a naturally-occurring wild-type SP1500 polypeptide sequence. For example, an SP1500 polypeptide may contain an amino acid sequence that is at least 60% or more (e.g. , at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 11 or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or more consecutive amino acids of the sequence shown in SEQ ID NO: 11). Alternatively, an SP1500 polypeptide may contain a portion (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or more consecutive amino acids) of a sequence that is at least 60% or more (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 11. In some embodiments, a nucleotide sequence encoding an SP1500 polypeptide is provided herein as SEQ ID NO: 18. In some embodiments, an SP 1500 polypeptide may comprises no more than 25 (including, e.g., no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2) amino acid modifications (e.g., deletion, substitution, and/or insertion) within the sequence of SEQ ID NO: 11 or a portion thereof as described herein. In some embodiments, such amino acid modifications may be present in the N-terminal portion and/or C- terminal portion.

[0113] In some embodiments, the disclosure includes nucleic acid sequences encoding any of the amino acids described herein. Due to degeneracy in the genetic code, those of ordinary skill in the art would understand that other DNA sequences (including codon-optimized sequences) could encode these polypeptides, as well as the others disclosed herein.

Fusion Proteins that Include Antigenic Polypeptides

[0114] Antigenic polypeptides described herein can be part of a fusion protein. For example, in some embodiments, an immunogenic complex described herein comprises a fusion protein that is or comprises a complementary affinity molecule and one or more antigenic polypeptides described herein. In some embodiments, a fusion protein of the immunogenic complex has carrier properties. In some embodiments, a fusion protein of the immunogenic complex has antigenic properties. In some embodiments, a fusion protein of the immunogenic complex has carrier properties and antigenic properties.

[0115] In some embodiments, the fusion protein of the immunogenic complex comprises one or more linkers and/or tags, e.g. , a histidine tag. In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence of GGGGSSS (SEQ ID NO:54), GGGGSGGGGSGGGGS (SEQ ID NO:58), or GGGGSGGGGSGGGGSM (SEQ ID NO:59). In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of GGGGSSS or GGGGSGGGGSGGGGSM. In some embodiments, the linker comprises the amino acid sequence AAA (SEQ ID NO:55). In some embodiments, the fusion protein of the immunogenic complex comprises a first linker comprising a polypeptide comprising the amino acid sequence of GGGGSSS, and a second linker comprising the amino acid sequence AAA. In some embodiments, the fusion protein of the immunogenic complex comprises a first linker comprising a polypeptide comprising the amino acid sequence of GGGGSGGGGSGGGGSM, and a second linker comprising the amino acid sequence AAA. In some embodiments, the fusion protein of the immunogenic complex comprises a first linker comprising a polypeptide comprising the amino acid sequence of GGGGSGGGGSGGGGSM, and a second linker comprising the amino acid sequence GGGGSSS. In some embodiments, such a linker may be synthesized, or derived from amino acid residues from a restriction site (e.g., a Not I restriction site).

Complementary Affinity Molecules

[0116] In some embodiments, a complementary affinity molecule comprises a biotin-binding moiety. In some embodiments, a fusion protein of the immunogenic complex comprises a biotin-binding moiety, and one or more polypeptide antigens. In some embodiments, a fusion protein comprises a biotin- binding moiety and two or more polypeptide antigens. As used herein, a “biotin-binding moiety” refers to a biotin-binding protein, a biotin-binding fragment thereof, or a biotin-binding domain thereof.

[0117] In some embodiments, MAPS complexes disclosed herein utilize the high affinity (dissociation constant [KD] ~ 10 ¹⁵M) non-covalent binding between biotin and rhizavidin, a biotin-binding protein that has no significant predicted homology with human proteins. Rhizavidin, a naturally occurring dimeric protein in the avidin protein family, was first discovered in Rhizobium etli, a symbiotic bacterium of the common bean. Rhizavidin has only a 22% amino acid identity with chicken avidin, a protein commonly found in eggs, but with high conservation of amino acid residues involved in biotin binding. No cross-reactivity to rhizavidin is observed in human serum samples obtained from subjects exposed to avidin (Helppolainen et al., Biochem J. 405:397- 405 (2007)), suggesting that rhizavidin antibodies may not cross-react with chicken avidin. Biotin conjugates have been used in several clinical applications without any reported adverse events (Buller et al, J Throb Haemost. 12:824-30 (2014); Paty et al, J Thromb Haemost. 8:722-9 (2010); Lazzeri et al, Eur J Nucl Med Mol Imaging. 31: 1505-11 (2004)).

[0118] In some embodiments, the biotin-binding moiety of the fusion protein comprises rhizavidin or a biotin-binding domain or biotin-binding fragment thereof, as further described in WO 2012/155053 the contents of which are herein incorporated by reference in their entirety. In some embodiments, a biotinbinding moiety is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to rhizavidin, or a biotin-binding domain or biotin-binding fragment thereof. In some embodiments, the biotin-binding moiety comprises a polypeptide of SEQ ID NO: 1 or a biotin-binding domain or biotin-binding fragment thereof (e.g., SEQ ID NO: 1 lacking 1, 2, 3, 4, 5, or more amino acids on the N- and/or C-terminus). In some embodiments, the biotin-binding moiety is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1, or biotin-binding domain or biotin-binding fragment thereof (e.g., lacking 1, 2, 3, 4, 5, or more amino acids on the N- and/or C-terminus). In some embodiments, the biotin-binding moiety comprises a polypeptide of SEQ ID NO: 2 or a biotin-binding domain or biotin-binding fragment thereof (e.g., SEQ ID NO: 2 lacking 1, 2, 3, 4, 5, or more amino acids on the N- and/or C-terminus). In some embodiments, the biotin-binding moiety is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 2, or biotin-binding domain or biotin-binding fragment thereof (e.g., lacking 1, 2, 3, 4, 5, or more amino acids on the N- and/or C- terminus). Antigenic Polysaccharides

[0119] In some embodiments, an antigenic polysaccharide is derived from an organism selected from the group consisting of: bacteria, archaea, viruses, or eukaryotic cells like fungi, insect, plant, or chimeras thereof. In some embodiments, the polysaccharide is derived from a pathogenic bacterium or virus. In some embodiments, the polysaccharide is or is derived from a glycoprotein. In specific embodiments, the polysaccharide is a pneumococcal capsular polysaccharide, a pneumococcal cell-wall polysaccharide, a meningococcal polysaccharide, a. Haemophilus influenze type b polysaccharide, a Streptococcus agalactiae polysaccharide, a Salmonella typhi Vi polysaccharide, a Klebsiella polysaccharide, a Pseudomonas polysaccharide, a Escherichia coli polysaccharide, or a Staphylococcus aureus polysaccharide.

[0120] In some embodiments, an antigenic polysaccharide is, or is derived from Gram-negative bacteria and/or Gram-positive bacteria. In some embodiments, an antigenic polysaccharide is, or is derived from one or more glycoproteins. In some embodiments, one or more such glycoproteins are, or are derived from one or more viruses. In some embodiments, an antigenic polysaccharide is, or is derived from .S', pneumoniae. In some embodiments antigenic polysaccharides included in an immunogenic composition described herein are, or are derived from one or more pathogens. In some embodiments, one or more antigenic polysaccharides are, or are derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 serotypes or strains (variants) of a pathogen. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 25 serotypes or strains (variants) of a pathogen, e.g., 26, 27, 28, 29, 30, 35, 40, 45, or 50 serotypes or strains. In some embodiments, one or more antigenic polysaccharides are, or are derived from more than 60, 70, 80, 90, or 100 serotypes or strains (variants) of a pathogen.

[0121] In some embodiments, the polysaccharide is a branched polysaccharide, or alternatively, can be a straight chain polysaccharide.

[0122] In some embodiments, an antigenic polysaccharide is a Vi antigen (Salmonella typhi capsular polysaccharide), pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharide, Hib (Haemophilus influenzae type B) capsular polysaccharide, meningococcal capsular polysaccharides, the polysaccharide of Bacillus anthracis (the causative agent of anthrax), and other bacterial capsular or cell wall polysaccharides, or any combinations thereof.

[0123] In some embodiments, the polysaccharide consists of or comprises a sugar moiety. For example, in some embodiments, a polysaccharide is a Vi polysaccharide of Salmonella typhi. The Vi capsular polysaccharide has been developed against bacterial enteric infections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis. 436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993). Vi is a polymer of a- 1 -^4-galacturonic acid with an N acetyl at position C-2 and variable O-acetylation at C-3. The virulence of S. typhi correlates with the expression of this molecule. Sharma et al., 101 PNAS 17492 (2004). The Vi polysaccharide vaccine of Salmonella typhi has several advantages: side effects are infrequent and mild, a single dose yields consistent immunogenicity and efficacy. Vi polysaccharide may be reliably standardized by physicochemical methods verified for other polysaccharide vaccines, Vi is stable at room temperature and it may be administered simultaneously with other vaccines without affecting immunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).

[0124] The polysaccharide can also be derived from Neisseria meningitidis, e.g., capsular polysaccharides from at least one, two, three or four of the serogroups A, C, W, W135, or Y. In some embodiments, the polysaccharide comprises Type 5, Type 8, or any of the polysaccharides or oligosaccharides of Staphylococcus aureus.

[0125] The polysaccharide can also be derived from Klebsiella pneumoniae, e.g., lipopolysaccharide (LPS)-derived polysaccharides or capsular polysaccharides. In some embodiments, LPS-derived polysaccharides are O polysaccharides (OPS). In some embodiments, LPS-derived polysaccharides are core O polysaccharides (COPS). In some embodiments, the polysaccharide is from, or derived from, an OPS from Klebsiella pneumoniae serotypes 01, 02, O2ac, 03, 04, 05, 07, 08, or 012. In some embodiments, the polysaccharide is from, or derived from, a CPS from Klebsiella pneumoniae KI, K2, K10, K16, or K19.

[0126] In some embodiments, the polysaccharide is from, or derived from, Pseudomonas aeruginosa, e.g., OPS, LPS, or exopolysaccharides. In some embodiments, the polysaccharide is from, or derived from, an OPS from ^Pseudomonas aeruginosa serotype selected from 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, Oi l, 012, 013, 014, 015, 016, 017, 018, 019, and 020. In some embodiments, the polysaccharide is, or is derived from, a capsular or capsular-like polysaccharide from Pseudomonas aeruginosa alginate, PsL, or Pel. In some embodiments, the polysaccharide is, or is derived from, an exopolysaccharide from Pseudomonas aeruginosa PsL.

[0127] In some embodiments, an immunogenic complex described herein includes one or more .S'. pneumoniae polysaccharides. In some embodiments, an immunogenic complex described herein includes one .S', pneumoniae polysaccharide. Capsular polysaccharides are used to distinguish serotypes of .S'. pneumoniae . There are at least 97 distinct serotypes of .S', pneumoniae polysaccharides, each having a different chemical structure. Figure 13 depicts exemplary structures and chemical information for certain .S', pneumoniae capsular polysaccharides. All structures are from European Pharmacopoeia 9.0. Serotype designations as used herein are designations according to Danish nomenclature (Kauffmann et al, Inti. Bull. Bact. Nomenclature and Taxonomy 10:31-41 (1960); Geno et al, Clin Microbiol Rev 28(3):871-899 (2015)). [0128] In some embodiments, an immunogenic complex includes one or more .S'. pneumoniae capsular polysaccharides from, or derived from, one or more .S'. pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, HA, 11B, 11C, HD, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 4 IF, 42, 43, 44, 45, 46, 47A, 47F, and 48.

[0129] In some embodiments, an immunogenic complex includes one or more .S'. pneumoniae capsular polysaccharides from, or derived from, one or more .S' pneumoniae serotypes selected from 1, 3, 4, 5 and 14.

[0130] In some embodiments, an immunogenic complex includes one .S', pneumoniae capsular polysaccharide from, or derived from, one .S', pneumoniae serotype. In some embodiments, an immunogenic complex includes one .S', pneumoniae capsular polysaccharide from, or derived from, one .S'. pneumoniae serotype selected from 1, 3, 4, 5, and 14.

[0131] In some embodiments, a polysaccharide is harvested and/or purified from a natural source; and in other embodiments, the polysaccharide is synthetic. Methods to produce synthetic polysaccharides are known to persons of ordinary skill and are encompassed in the compositions and methods as disclosed herein.

Methods of Isolating and Purifying Polysaccharides

[0132] In some embodiments, the disclosure provides methods of purifying one or more polysaccharides described herein from one or more cellular components of bacteria. In some embodiments, methods comprise purifying capsular polysaccharides from one or more cellular components of bacteria.

[0133] In some embodiments, the bacteria are Gram-negative. In some embodiments, the bacteria are Gram -positive. In some embodiments, the bacteria are .S', pneumoniae.

[0134] In some embodiments, the cellular components include protein. In some embodiments, the cellular proteins include nucleic acid. In some embodiments, the cellular components include lipids. In some embodiments, the cellular components include polysaccharides. In some embodiments, the cellular components are part of a lysate.

[0135] In some embodiments, the polysaccharide purification processes incorporate a series of ethanol precipitations, washes of crude polysaccharide preparations with ethanol, diethyl ether, and/or acetone, and drying under vacuum to furnish purified products. In some embodiments, a phenol extraction step is incorporated for polysaccharide purifications. In some embodiments, the purification process employs a CTAB (cetyltrimethyl ammonium bromide) precipitation step in addition to using ethanol and phenol precipitation steps.

Methods of Biotinylating Polysaccharides

[0136] In some embodiments, the disclosure provides methods of biotinylating one or more polysaccharides described herein. In some embodiments, the method comprises reacting purified polysaccharides with l-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of hydroxyl groups in the polysaccharides followed by the addition of amine PEG biotin under conditions that result in covalent linkage of biotin to the polysaccharides. In some embodiments, the desired level of biotinylation is achieved by varying the ratio of CDAP to polysaccharide. In some embodiments, the biotinylated polysaccharides are purified by filtration to remove process residuals such as unreacted biotin, dimethylaminopyridine, acetonitrile, cyanide and unreacted glycine. In some embodiments, the level of polysaccharide biotinylation described herein is optimized to reduce the amount of accessible biotin following MAPS complexation.

Immunogenic Conjugates

[0137] In some embodiments, the disclosure includes immunogenic conjugates that include (i) one or more polypeptides (e.g., antigenic polypeptides) described herein conjugated to (ii) one or more polysaccharides described herein. In some embodiments, one or more conjugated polysaccharides comprise a capsular polysaccharide of .S', pneumoniae . In some embodiments, one or more polypeptides of an immunogenic conjugate comprise an antigenic polypeptide of .S', pneumoniae . In some embodiments, an antigenic polypeptide of an immunogenic conjugate is or comprises a fusion protein.

[0138] As disclosed herein, one or more antigenic polypeptides of the immunogenic conjugate comprise a polypeptide antigen, or a fusion protein comprising one or more polypeptide antigens, that is expressed by the same pathogen as the polysaccharide antigen of the immunogenic conjugate. In some embodiments, the immunogenic conjugate comprises one or more antigenic polypeptides covalently associated with one or more antigenic polysaccharides, where the one or more antigenic polypeptides are expressed by the same pathogen from which the antigenic polysaccharide was derived, including, in some embodiments, where the one or more antigenic polypeptide are expressed by the same serotype (or variant) of the pathogen from which the antigenic polysaccharide was derived.

Manufacture of Immunogenic Complexes and Immunogenic Conjugates

[0139] The present disclosure includes methods for manufacturing immunogenic complexes described herein. In some embodiments, a method of manufacturing an immunogenic complex comprises complexing at least one biotinylated polysaccharide (e.g., a biotinylated polysaccharide described herein) with at least one biotin-binding fusion protein described herein. In some embodiments, the present disclosure includes methods for manufacturing immunogenic conjugates described herein. In some embodiments, a method of manufacturing an immunogenic conjugate comprises conjugating at least one polysaccharide (e.g., polysaccharide described herein) with at least one polypeptide described herein.

[0140] In some embodiments, the average (e.g., the mean) protein (e.g., antigenic protein) to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, or 10: 1 (weight/weight [w/w]). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 1: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 2: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 3: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 4: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 5: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 6: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 7: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 8: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 9: 1 (w/w). In some embodiments, the average protein to polysaccharide ratio of a plurality of immunogenic complexes or immunogenic conjugates is approximately 10: 1 (w/w). Immunogenic compositions and vaccines of the invention may comprise mixtures of immunogenic complexes or immunogenic conjugates with different average protein to polysaccharide ratios.

[0141] In some embodiments, an immunogenic composition (e.g., a vaccine) comprises a plurality of immunogenic complexes comprising a fusion protein described herein and a capsular polysaccharide. In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1,7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, or 10: 1 (weight/weight [w/w]). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 1: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 2: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 3: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 4: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 5: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 6: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 7: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 8: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 9: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is approximately 10: 1 (w/w). In some embodiments, the average ratio of fusion protein to capsular polysaccharide in the plurality of immunogenic complexes is chosen to enhance the polysaccharide immunogenicity potential (carrier or presentation function) and/or to elicit protection against, or to inhibit, pneumococcal colonization by any pneumococcus (independent of polysaccharide serotype) through a protein-specific immune response. Immunogenic compositions (e.g., vaccines) of the present disclosure may comprise mixtures of immunogenic complexes with different average protein to polysaccharide ratios.

Immunogenic and Vaccine Compositions

[0142] Another aspect of the disclosure provides compositions that include one or more immunogenic complexes described herein. For example, an immunogenic composition, e.g., vaccine composition, can include one or more immunogenic complexes described herein. In some embodiments, such compositions can include a plurality of one type of immunogenic complex described herein. For example, a composition can include a population of one type of immunogenic complex, where all of the immunogenic complexes include the same antigenic polypeptide and the same antigenic polysaccharide. Additionally or alternatively, such compositions can include a plurality of more than one type of immunogenic complex described herein. For example, a composition can include populations of different types of immunogenic complexes. In some embodiments, a composition can include a population of a first type of immunogenic complex and a population of a second type of immunogenic complex, where the first type and the second type of the immunogenic complex have different antigenic polypeptides and/or different antigenic polysaccharides. In some embodiments, a composition can include a population of a first type of immunogenic complex and a population of a second type of immunogenic complex, where the first type and the second type of the immunogenic complex include the same antigenic polypeptide and different antigenic polysaccharides (e.g., polysaccharides of different serotypes). In some embodiments, immunogenic complexes described herein are formulated into a pharmaceutical composition. In some embodiments, a pharmaceutical composition may be a vaccine. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an adjuvant.

Vaccine compositions

[0143] In some embodiments, a vaccine composition is a monovalent vaccine. In some embodiments, a vaccine composition is a polyvalent or multivalent vaccine. In some embodiments, a vaccine composition is a mono variant vaccine, comprising one or more antigens from one strain or variant of a pathogen. In some embodiments, a vaccine composition is a multivariant vaccine, comprising one or more antigens from more than one strain or variant of a pathogen. In some embodiments, the valency of a vaccine composition refers to the number of species of immunogenic complexes present in the vaccine composition. The valency of a vaccine described herein is not limiting with respect to the total antigens present in said pharmaceutical composition, immunogenic complex, or vaccine, or to the number of pathogen strains for which administration of said pharmaceutical composition, immunogenic complex, immunogenic composition, or vaccine composition may induce an immune -protective response.

[0144] In some embodiments, a vaccine composition comprises between 1-50 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-40 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-35 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1- 30 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-30 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-24 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-15 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-9 species of immunogenic complexes. In some embodiments, a vaccine composition comprises between 1-5 species of immunogenic complexes. In some embodiments, a vaccine is a polyvalent vaccine.

[0145] In some embodiments, a vaccine 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, or 50 species of immunogenic complexes.

[0146] In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g. , in immunogenic compositions) in amounts such that the weight of polysaccharides in the vaccine composition from each immunogenic complex is about the same, e.g., present in a w/w ratio of about 1: 1. In some embodiments, the weight of polysaccharides in the vaccine contributed by each immunogenic complex is about 0.20 pg, about 0.25 pg, about 0.5 pg, about 1 pg, about 1.5 pg, about 2 pg, about 2.5 pg, about 3 pg, about 3.5 pg, about 4 pg, about 4.5 pg, about 5 pg, about 5.5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, or about 12 pg. In some embodiments, the weight of polysaccharides in the vaccine contributed by each immunogenic complex is more than 12 pg, e.g., 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, or more.

[0147] In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g. , in immunogenic compositions) in amounts such that the weight of polysaccharides in the vaccine composition contributed by each immunogenic complex is different, e.g., present in a w/w ratio that is not about 1: 1. In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g., in immunogenic compositions) in amounts such that the weight of polysaccharide in the vaccine composition contributed by a first immunogenic complex and a second immunogenic complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10. In some embodiments, the vaccine composition comprises a mixture of immunogenic complexes, such that the weight of polysaccharide in a vaccine contributed by an immunogenic complex ranges from about 0.20 pg to about 6 pg. In some embodiments, the vaccine composition comprises a mixture of immunogenic complexes, such that the weight of polysaccharide in a vaccine contributed by an immunogenic complex ranges from about 0.20 pg to about 12 pg. In some embodiments, the vaccine composition comprises a mixture of immunogenic complexes, such that the weight of polysaccharides in the vaccine contributed by each immunogenic complex ranges from about 0.20 pg to about 20 pg. In some embodiments, the vaccine composition comprises a mixture of immunogenic complexes, such that the weight of polysaccharides in the vaccine contributed by each immunogenic complex ranges from about 0.20 pg to about 40 pg.

[0148] In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g., in immunogenic compositions) in amounts such that the combined weight of polysaccharides and polypeptides in the vaccine composition contributed by each immunogenic complex (e.g., in an immunogenic composition) is about the same, e.g., present in a w/w proteimPS ratio of about 1: 1. In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g., in immunogenic compositions) in amounts such that the combined weight of polysaccharides and polypeptides in the vaccine composition contributed by each immunogenic complex (e.g., in an immunogenic composition) is present in a w/w proteimPS ratio of about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, or about 10: 1.

[0149] In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine contributed by each immunogenic complex is about 0.20 pg, about 0.40 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 14 pg, about 16 pg, about 18 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, or about 110 pg. [0150] In some embodiments, a vaccine composition comprises two or more species of immunogenic complexes (e.g., in immunogenic compositions) in amounts such that the combined weight of polysaccharides and polypeptides in the vaccine composition contributed by each immunogenic complex is different, e.g. , present in a w/w proteimPS ratio that is not about 1: 1, e.g. , a proteimPS ratio that is 2: 1, 3: 1, 4: 1. 5: 1. 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. In some embodiments, the vaccine composition comprises a mixture of immunogenic complexes, such that the combined weight of polysaccharides and polypeptides in the vaccine contributed by each immunogenic complex ranges from about 0.4 pg to about 110 pg.

Uses of Immunogenic and Vaccine Compositions

[0151] In some embodiments, an immunogenic complex described herein that includes one or more antigenic polysaccharides is characterized in that one or more of the opsonization potential, or immune response to one or more antigenic polysaccharides is increased relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, one or more of the opsonization potential, immune response to the one or more antigenic polysaccharides is increased at least 1-fold, 2-fold, 3 -fold, 4-fold, or 5 -fold relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, the predetermined level is a pre-immune level. In some embodiments, the predetermined level is a pre-immune level.

[0152] In some embodiments, an immune response is compared to a control composition. In any of the embodiments described herein, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition). In any of the embodiments described herein, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition). In any of the embodiments described herein, a control composition may comprise an adjuvant present in the immunogenic composition (or vaccine composition), and not comprise an antigenic polysaccharide and/or an immunogenic polypeptide present in the immunogenic composition (or vaccine composition). In any of the embodiments described herein, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) that is not associated with a polypeptide antigen. In any of the embodiments described herein, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) that is covalently conjugated to a polypeptide antigen and/or a carrier polypeptide. In any of the embodiments described herein, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) and not comprise a polypeptide antigen expressed by a pathogen (e.g., a control composition is a conjugate vaccine comprising a CRM197 carrier protein, e.g., PCV20). [0153] In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces an immune response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces an immune response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces a protective immune response.

[0154] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or a CD4-/CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0155] In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces antibody production against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an immune response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic complex described herein, upon administration to a subject, induces a protective immune response.

[0156] In therapeutic embodiments, the vaccine is administered to a subject post-infection. The vaccine may be administered shortly after infection, e.g. before symptoms or sequelae manifest, or may be administered during or after manifestation of symptoms or sequelae.

[0157] In some embodiments, the vaccine compositions of the invention confer protective immunity, allowing a vaccinated subject to exhibit delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, as the result of his or her exposure to the vaccine. In certain embodiments, the reduction in severity of symptoms or sequelae is at least 25%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., relative to a control (e.g., control composition). Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. In particular, cellular immunity may be mediated by TH 1 or TH 17 cells.

[0158] In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or CD4-/CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0159] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an antibody or B cell response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0160] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces a T cell response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, the immune response is a protective immune response. In some embodiments, the immune response comprises neutralizing antibodies.

[0161] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an immune response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces a protective immune response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0162] In some embodiments, the subject is a human. In some embodiments, the human is between about 2 weeks of age and about 6 weeks of age. In some embodiments, the human is between about 6 weeks of age and about 6 years of age. In some embodiments, the human is between about 6 years of age and about 18 years of age. In some embodiments, the human is between about 18 years of age and about 50 years of age. In some embodiments, the human is about 50 years of age and about 75 years of age. In some embodiments, the human is about 75 years of age or older.

[0163] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an immune response against one or more pathogen strains at a level greater than a control composition. In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including THI, TH2, or TH17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or CD4-/CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response, and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response. In some embodiments, the immune response is a protective immune response. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0164] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces a T cell response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces a T cell response against one or more pathogens in the subject at level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0165] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an antibody or B cell response against one or more pathogens in the subject at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about

19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about

80%, about 85%, about 90%, or about 95% of the control composition.

[0166] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, induces an antibody or B cell response against one or more polysaccharides (included in the immunogenic composition or vaccine) in the subject at level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.

[0167] In some embodiments, an immunogenic composition or vaccine described herein, upon administration to a subject, potentiates a B cell recall response to a polysaccharide antigen of a pathogen, e.g., to a predetermined target level. For example, in some embodiments, an immunogenic composition is administered to a subject, and following subsequent exposure of the subject to the pathogen, the subject exhibits a potentiated B cell recall response (mediated by the prior administration of the immunogenic composition) to the polysaccharide antigen, to the predetermined target. In some embodiments, the predetermined target level is a level that is higher (e.g., about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% higher) than a control level (e.g., a level of a B cell recall response induced in a subject following administration of a control composition). In some embodiments, the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or by killing of the pathogen by immune sera from the subject in an opsonophagocytic assay (OPA), at a level that is at least 20% higher (e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% higher) than a control level (e.g., a level of a B cell recall response induced in a subject following administration of a control composition). In some embodiments, the predetermined target level is determined based on a corresponding level of a B cell recall response induced in a non-human mammalian model upon administration of the immunogenic composition to the non-human mammalian model and subsequent exposure of the non-human mammalian model to the pathogen. In some embodiments, a B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells. In some embodiments, a B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polypeptide antigen-specific Th cells. In some embodiments, a B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells and polypeptide antigen-specific Th cells.

[0168] In some embodiments, an immunogenic composition described herein is administered to a subject to immunize the subject against a pathogen. In some embodiments, the administered dose is lower (e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% lower) than that of a control composition to achieve in the subject, upon exposure to the pathogen, an equivalent or greater B cell recall response to a polysaccharide antigen included in the immunogenic composition. In some embodiments, the administered dose provides protection against the pathogen for a longer period of time (e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, about 100%, about 150%, about 200%, about 500%, or longer) than provided by the same dose of a control composition. In some embodiments, the protection against the pathogen comprises a Thl response. In some embodiments, the Thl response comprises production of IFN-y and/or TNF-a by CD4+ T cells at a level that is at least 1.1-fold higher (e.g., at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, 10-fold, or higher) than a corresponding level of IFN-y and/or TNF-a produced by CD4+ T cells upon administration to the subject of a control composition. In some embodiments, the protection against the pathogen comprises a Thl7 response. In some embodiments, the Thl7 response comprises production of IL- 17, IL-21, IL-22, IL24 and/or IL-26 by CD4+ T cells at a level that is at least 1.1 -fold higher (e.g., at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or higher) than a corresponding level of IL- 17, IL-21, IL-22, IL24 and/or IL-26 produced by CD4+ T cells upon administration to the subject of the equivalent dose of a control composition. In some embodiments, the protection against the pathogen comprises a CD8 response. In some embodiments, the CD8 response comprises production of IFN-y, granzyme B, and/or perforin by CD8 T cells at a level that is at least 1.1-fold higher (e.g., at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or higher) than a corresponding level of IFN-y, granzyme B, and/or perforin produced by CD8 T cells upon administration to the subject of the equivalent dose of a control composition.

[0169] In some embodiments, an immunogenic composition described herein (e.g., a MAPS vaccine) is administered to a subject (who has received an initial (prime) vaccine against a pathogen) as a booster vaccine. In some embodiments, the booster MAPS vaccine, upon administration to the subject who has received the prime vaccine, induces a B cell response to the polysaccharide antigen at a predetermined target level. In some embodiments, the predetermined target level is, e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% higher than a corresponding level of B cell response in a subject who has received a booster vaccine comprising a control composition (e.g., a control composition comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein).

Antibody Compositions

[0170] Some embodiments provide for an antibody composition comprising antibodies raised in a mammal immunized with an immunogenic complex of the invention. In some embodiments, an antibody comprises at least one antibody selected from the group consisting of mAbs and anti-idiotype antibodies. In some embodiments, an antibody composition comprises neutralizing antibodies. In some embodiments, an antibody composition comprises an isolated gamma globulin fraction. In some embodiments, an antibody composition comprises polyclonal antibodies. In some embodiments, the antibody composition is administered to a subject. In some embodiments, the antibody composition administered to a subject confers passive immunization.

Vaccine Formulations

[0171] Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects can receive one or several booster immunizations adequately spaced in time.

[0172] The immunogenic complexes described herein, and/or preparations thereof may be formulated in a unit dosage form for ease of administration and uniformity of dosage. The specific therapeutically effective dose level for any particular subject or organism may depend upon a variety of factors including the severity or degree of risk of infection; the activity of the specific vaccine or vaccine composition employed; other characteristics of the specific vaccine or vaccine composition employed; the age, body weight, general health, sex of the subject, diet of the subject, pharmacokinetic condition of the subject, the time of administration (e.g. , with regard to other activities of the subject such as eating, sleeping, receiving other medicines including other vaccine doses, etc.), route of administration, rate of excretion of the specific vaccine or vaccine composition employed; vaccines used in combination or coincidental with the vaccine composition employed; and like factors well known in the medical arts.

[0173] Immunogenic complexes for use in accordance with the present disclosure may be formulated into compositions (e.g., pharmaceutical compositions) according to known techniques. Vaccine preparation is generally described in Vaccine Design (Powell and Newman, 1995). For example, an immunologically amount of a vaccine product can be formulated together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials. [0174] In general, pharmaceutically acceptable carrier(s) include solvents, dispersion media, and the like, which are compatible with pharmaceutical administration. For example, materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose, dextrose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; polyols such as glycerol, propylene glycol, and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator (Martin, 1975).

[0175] Vaccines may be formulated by combining one or more of the immunogenic complexes disclosed herein with carriers and/or other optional components by any available means including, for example, conventional mixing, granulating, dissolving, lyophilizing, or similar processes.

[0176] Vaccine compositions useful in the provided methods may be lyophilized up until they are about to be used, at which point they are extemporaneously reconstituted with diluent. In some embodiments, vaccine components or compositions are lyophilized in the presence of one or more other components (e.g., adjuvants), and are extemporaneously reconstituted with saline solution. Alternatively, individual components, or sets of components may be separately lyophilized and/or stored (e.g., in a vaccination kit), the components being reconstituted and either mixed prior to use or administered separately to the subject.

[0177] Lyophilization can produce a more stable composition (for instance by preventing or reducing breakdown of polysaccharide antigens). Lyophilizing of vaccines or vaccine components is well known in the art. Typically, a liquid vaccine or vaccine component is freeze dried, often in the presence of an anti-caking agent (such as, for example, sugars such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10-200 mg/ml. Lyophilization typically occurs over a series of steps, for instance a cycle starting at -69° C, gradually adjusting to -24°C over 3 h, then retaining this temperature for 18 h, then gradually adjusting to -16°C over 1 h, then retaining this temperature for 6 h, then gradually adjusting to +34°C over 3 h, and finally retaining this temperature over 9 h.

[0178] In some embodiments, a vaccine is a liquid. In some embodiments, the liquid is a reconstituted lyophylate. In some embodiments, a vaccine has a pH of about 5, about 6, about 7, or about 8. In some embodiments, a vaccine has a pH between about 5 and about 7.5. In some embodiments, a vaccine has a pH between 5 and 7.5. In some embodiments, a vaccine has a pH between about 5.3 and about 6.3. In some embodiments, a vaccine has a pH between 5.3 and 6.3. In some embodiments a vaccine has a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about

5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about

6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0179] Vaccines or vaccine components for use in accordance with the present invention may be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or immune-stimulating complexes (ISCOMs).

[0180] In certain situations, it may be desirable to prolong the effect of a vaccine or for use in accordance with the present invention, for example by slowing the absorption of one or more vaccine components. Such delay of absorption may be accomplished, for example, by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the product then depends upon its rate of dissolution, which in turn, may depend upon size and form. Alternatively, or additionally, delayed absorption may be accomplished by dissolving or suspending one or more vaccine components in an oil vehicle. Injectable depot forms can also be employed to delay absorption. Such depot forms can be prepared by forming microcapsule matrices of one or more vaccine components a biodegradable polymers network. Depending upon the ratio of polymer to vaccine component, and the nature of the particular polymer(s) employed, the rate of release can be controlled.

[0181] Examples of biodegradable polymers that can be employed in accordance with the present invention include, for example, poly (orthoesters) and poly(anhydrides). One particular exemplary polymer is polylactide -polyglycolide.

[0182] Depot injectable formulations may also be prepared by entrapping the product in liposomes or microemulsions, which are compatible with body tissues.

[0183] Polymeric delivery systems can also be employed in non-depot formulations including, for example, oral formulations. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, etc., can be used in oral formulations. Polysaccharide antigens or conjugates may be formulated with such polymers, for example to prepare particles, microparticles, extrudates, solid dispersions, admixtures, or other combinations in order to facilitate preparation of useful formulations (e.g., oral).

[0184] Vaccines for use in accordance with the present invention include immunogenic compositions, and may additionally include one or more additional active agents (i. e. , agents that exert a biological effect - not inert ingredients). For example, it is common in vaccine preparation to include one or more adjuvants. It will be appreciated that such additional agents may be formulated together with one or more other vaccine components, or may be maintained separately and combined at or near the time of administration. In some embodiments, such additional components may be administered separately from some or all of the other vaccine components, within an appropriate time window for the relevant effect to be achieved.

Adjuvants

[0185] The vaccine formulations and immunogenic compositions described herein may include an adjuvant. Adjuvants, generally, are agents that enhance the immune response to an antigen. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et al, 2003). In most vaccine formulations, the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen. Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and/or matrices, and liposomes. In contrast, immunostimulatory adjuvants are sometimes from or derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), or CpG-containing DNA, which activate cells of the innate immune system.

[0186] Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. Non-limiting examples of organic adjuvants include cholera toxin/toxoids, other enterotoxins/toxoids or labile toxins/toxoids of Gram-negative bacteria, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0187] Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the generation, proliferation, or activation of THI cells or TH2 cells. In other embodiments, the adjuvant induces the generation, proliferation, or activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.

[0188] In certain embodiments, the adjuvant induces the generation, proliferation, or activation of TH17 cells. The adjuvant may promote the CD4+ or CD8+ T cells to secrete IL-17. In some embodiments, an adjuvant that induces the generation, proliferation, or activation of TH 17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL- 17 levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce TH17 generation, proliferation, or activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the generation, proliferation, or activation of TH 17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL-17 may be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli, or ETEC), activate a TH 17 response. Thus, in some embodiments, the adjuvant is a toxin or toxoid. Mutant derivates of labile toxin (toxoids) that are active as adjuvants but significantly less toxic can be used as well. Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP- ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al, 1995) and LTK63 (Williams et al, 2004), LT-G192 (Douce et al, 1999), and LTR72 (Giuliani et al, 1998).

[0189] In some embodiments, the adjuvant comprises a VLP (virus-like particle). One such adjuvant platform, Alphavirus replicons, induces the activation of TH17 cells using alphavirus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL- 17 or a cytokine that stimulates IL- 17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, a vaccine formulation is administered to a subject in combination with a nucleic acid encoding a cytokine.

[0190] Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a TH 17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram -negative bacteria, such as .S', minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans et al, 2003. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka et al, 2007). Various TLR agonists (including TLR-4 agonists) have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).

[0191] In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.

[0192] In certain embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.

[0193] Adjuvants may be covalently or non-covalently bound to antigens. In some embodiments, the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen- presenting cells (APCs). In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (e.g., see Wu et al, 2005).

[0194] In some embodiments, immunogenic complexes described herein are formulated and/or administered in combination with an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, and phosphate aluminum hydroxide. In some embodiments, the adjuvant comprises aluminum phosphate. In some embodiments, the adjuvant is aluminum phosphate.

[0195] Typically, the same adjuvant or mixture of adjuvants is present in each dose of a vaccine. Optionally, however, an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (i.e., booster shots). Alternatively, a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human subjects, non-human animals, or both.

[0196] Vaccines for use in accordance with the present invention may include, or be administered concurrently with, other antimicrobial, antiviral, or anti-inflammatory therapies. For example, such vaccines may include or be administered with one or more agents that kills or retards growth of a pathogen. Such agents include, for example, remdesivir, lopinavir and/or ritonavir (e.g., Kaletra), oseltamivir (Tamiflu), favipiravir, umifenovir, galidesivir, dexamethasone, colchicine, convalescent plasma, monoclonal antibodies (e.g., one or more of bamlanivimab, LY-C0VOI6, etesevimab, casirivimab, indevimab, sarilumab, tocilizumab), IL-6 inhibitors, kinase inhibitors, interferons, penicillin, vancomycin, erythromycin, azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.

[0197] Alternatively or additionally, vaccines for use in accordance with the present invention may include, or be administered with, one or more other vaccines or therapies. For example, one or more non-SARS-CoV-2antigens may be included in or administered with the vaccines.

Additional Components and Excipients

[0198] In addition to the antigens and the adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components.

[0199] In certain embodiments, the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L- histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.

[0200] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.

[0201] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more surfactants, for example, but not limited to, polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20), Polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100), and 4- (l,l,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or non-ionic.

[0202] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

[0203] In certain embodiments, a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multidose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid. Methods of Administration

[0204] Any effective route of administration of an immunogenic composition described herein may be utilized such as, for example, oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, transdermal, intradermal, rectal, vaginal, topical, ocular, pulmonary, or by contact application. In some embodiments, vaccine compositions may be injected (e.g., via intramuscular, intraperitoneal, intradermal and/or subcutaneous routes); or delivered via the mucosa (e.g., to the oral/alimentary, respiratory, and/or genitourinary tracts). In some embodiments, method to deliver an immunogenic composition or vaccine as disclosed herein is deliverered to a mucosal surface within the oral or nasal cavity of a subject. Methods and devices useful for such mucosal delivery are disclosed in US patents 10,286,164, 6,012,454, W02000/051672, WO1998053869, W02002/068030, WO2008/122795, USD723156S1, USD725769, US20160310683, each of which are incorporated herein in its entiriy by reference. In some embodiments of the invention, it may be desirable to administer different doses of a vaccine by different routes; in some embodiments, it may be desirable to administer different components of one dose via different routes. In some embodiments, an immunogenic composition or vaccine disclosed herein is administered intramuscularly. In some embodiments, an immunogenic composition or vaccine disclosed herein is administered subcutaneously.

[0205] In some embodiments of the present invention, pharmaceutical compositions (e.g., vaccines) are administered intradermally. Conventional technique of intradermal injection, the "Mantoux procedure", comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26-31 gauge) facing upwards the needle is inserted at an angle of between 10- 15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced while providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.

[0206] Devices that are specifically designed to administer liquid agents into or across the skin have been described, for example the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described for example in WO 01/13977; US Patent No. 5,480,381, US Patent No. 5,599,302, US Patent No. 5,334,144, US Patent No. 5,993,412, US Patent No. 5,649,912, US Patent No.

5,569,189, US Patent No. 5,704,911, US Patent No. 5,383,851, US Patent No. 5,893,397, US Patent No.

5,466,220, US Patent No. 5,339,163, US Patent No. 5,312,335, US Patent No. 5,503,627, US Patent No.

5,064,413, US Patent No. 5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US Patent No.

4,941,880, US Patent No. 4,940,460, WO 97/37705 and WO 97/13537. Other methods of intradermal administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or applied to the surface of the skin (transdermal or transcutaneous delivery WO 98/20734; WO 98/28037). [0207] As described above, pharmaceutical compositions (e.g, vaccines) may be administered as a single dose or as multiple doses. It will be appreciated that an administration is a single “dose” so long as all relevant components are administered to a subject within a window of time; it is not necessary that every component be present in a single composition. For example, administration of two different immunogenic compositions, within a period of less than 24 h, is considered a single dose. To give but one example, immunogenic compositions having different antigenic components may be administered in separate compositions, but as part of a single dose. As noted above, such separate compositions may be administered via different routes or via the same route. Alternatively or additionally, in embodiments wherein a vaccine comprises a combination of immunogenic compositions and additional types of active agents, immunogenic compositions may be administered via one route, and a second active agent may be administered by the same route or by a different route.

[0208] Pharmaceutical compositions (e.g., vaccines) are administered in such amounts and for such time as is necessary to achieve a desired result. In certain embodiments of the present invention, a vaccine composition comprises an immunologically effective amount of at least immunogenic composition. The exact amount required to achieve an immunologically effective amount may vary, depending on the immunogenic composition, and from subject to subject, depending on the species, age, and general condition of the subject, the stage of the disease, the particular pharmaceutical mixture, its mode of administration, and the like.

[0209] The amount of polypeptide antigen(s), polysaccharide antigen(s) or conjugate(s) in each pharmaceutical composition (e.g., vaccine) dose is selected to allow the vaccine, when administered as described herein, to induce an appropriate immune-protective response without significant, adverse side effects.

[0210] In some embodiments, administration of a vaccine (e.g., a vaccine composition) described herein may involve the delivery of a single dose. In some embodiments, administration may involve an initial dose followed by one or several additional immunization doses, adequately spaced. An immunization schedule or regimen is a program for the administration of one or more specified doses of one or more specified vaccines, by one or more specified routes of administration, at one or more specified ages of a subject.

[0211] Immunization schedules of the present disclosure are provided to induce an immune response (e.g., an immunoprotective response) in a subject sufficient to reduce at least one measure selected from the group consisting of incidence, prevalence, frequency, and/or severity of at least one infection, disease, or disorder, and/or at least one surrogate marker of the infection, disease, or disorder, in a population and/or subpopulation of the subject(s). A supplemental immunization schedule is one which has this effect relative to the standard schedule which it supplements. A supplemental schedule may call for additional administrations and/or supra-immunogenic doses of the immunogenic compositions disclosed herein, found in the standard schedule, or for the administration of vaccines not part of the standard schedule. A full immunization schedule of the present invention may comprise both a standard schedule and a supplemental schedule. Exemplary sample vaccination schedules are provided for illustrative purposes. Detailed descriptions of methods to assess immunogenic response discussed herein allow one to develop alterations to the sample immunization schedules without undue experimentation.

[0212] In some embodiments of the present disclosure, a first administration of a vaccine occurs when a subject is more than about 2 weeks old, more than about 5 weeks old, more than about 1 year old, more than about 2 years old, more than about 15 years old, or more than about 18 years old.

[0213] In some embodiments, a first administration of a vaccine occurs when a subject is about two months old. In some embodiments, a second administration of a vaccine occurs when a subject is about four months old. In some embodiments, a third administration of a vaccine occurs when a subject is about six months old. In some embodiments, a fourth administration of a vaccine occurs when a subject is between about twelve months old and about fifteen months old.

[0214] In some embodiments of the present disclosure, a first administration of a vaccine occurs when a subject is more than about 18 years old, more than about 50 years old, more than about 55 years old, more than about 60 years old, more than about 65 years old, or more than about 70 years old.

[0215] In some embodiments of the disclosure, a single administration of vaccine is employed. It is possible that the purposes of the present invention can be served with a single administration, especially when one or more utilized vaccine polypeptide(s), polysaccharide (s) and/or immunogenic complex(es) or combinations thereof is/are strong, and in such a situation a single dose schedule is sufficient to induce a lasting immune -protective response.

[0216] In certain embodiments, it is desirable to administer two or more doses of vaccine, for greater immune-protective efficacy and coverage. Thus, in some embodiments, a number of doses is at least two, at least three or more doses. There is no set maximum number of doses, however it is good clinical practice not to immunize more often than necessary to achieve the desired effect.

[0217] Without being bound by theory, a first dose of vaccine administered according to the disclosure may be considered a “priming” dose. In certain embodiments, more than one dose is included in an immunization schedule. In such a scenario, a subsequent dose may be considered a “boosting” dose.

[0218] A priming dose may be administered to a naive subject (a subject who has never previously received a vaccine). In some embodiments, a priming dose may be administered to a subject who has previously received a vaccine at least five or more years previous to administration of an initial vaccine dose according to the invention. In other embodiments, a priming dose may be administered to a subject who has previously received a vaccine at least twenty or more years previous to administration of a priming vaccine according to the disclosure.

[0219] When an immunization schedule calls for two or more separate doses, the interval between doses is considered. The interval between two successive doses may be the same throughout an immunization schedule, or it may change as the subject ages. In immunization schedules of the present invention, once a first vaccine dose has been administered, there is a first interval before administration of a subsequent dose. A first interval is generally at least about 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 12 months, or longer. Where more than one subsequent dose(s) are administered, second (or higher) intervals may be provided between such subsequent doses. In some embodiments, all intervals between subsequent doses are of the same length; in other embodiments, second intervals may vary in length. In some embodiments, the interval between subsequent doses may be at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months or at least about 2 years. In certain embodiments, the interval between doses may be up to 3 years, up to about 4 years, or up to about 5 years or 10 years or more. In certain embodiments, intervals between subsequent doses may decrease as the subject ages.

[0220] It will be appreciated by those skilled in the art that a variety of possible combinations and sub-combinations of the various conditions of timing of the first administration, shortest interval, largest interval and total number of administrations (in absolute terms, or within a stated period) exist, and all of these combinations and sub-combinations should be considered to be within the inventor's contemplation though not explicitly enumerated here.

Assays for Determining Immune Response

[0221] In some embodiments, a method of assessing the immunogenicity of an immunogenic composition (and/or vaccine composition) described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by opsonophagocytic killing assay (OPK, OPA), plaque reduction neutralization test (PRNT), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition (or vaccine composition), and not comprise an antigenic polysaccharide and/or an immunogenic polypeptide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) that is not associated with a polypeptide antigen. In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) that is covalently conjugated to a polypeptide antigen and/or a carrier polypeptide.

[0222] In some embodiments, a method of assessing the potency of an immunogenic composition (or vaccine composition) described herein comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, TH1/TH17 cell response, cytokine level measurement and functional antibody levels as measured by OPK (OPA), plaque reduction neutralization test (PRNT), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of disease. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) and not comprise an antigenic polysaccharide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition (or vaccine composition), and not comprise an antigenic polysaccharide and/or an immunogenic polypeptide present in the immunogenic composition (or vaccine composition). In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) that is not associated with a polypeptide antigen. In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition (or vaccine composition) that is covalently conjugated to a polypeptide antigen and/or a carrier polypeptide.

[0223] Generally speaking, it may be desirable to assess humoral responses, cellular responses, and/or interactions between the two. Where humoral responses are being assessed, antibody titers and/or types (e.g., total IgG, IgGl, IgG2, IgM, IgA, etc.) to specific pathogen polysaccharides or polypeptides (either serotype-specific or conserved across two or more serotypes) may be determined, for example before and/or after administration of an initial or a boosting dose of vaccine (and/or as compared with antibody levels in the absence of antigenic stimulation). Cellular responses may be assessed by monitoring reactions such as delayed type hypersensitivity responses, etc. to the carrier protein. Cellular responses can also be measured directly by evaluating the response of peripheral blood mononuclear cells (PBMCs) monocytes to stimulation with the antigens of interest. Precursor and memory B cell populations may be assessed in enzyme linked immunospot (ELISpot) assays directed against specific pathogen polysaccharides or polypeptides.

[0224] Any of a variety of assays may be employed to detect levels and/or activity of antibodies in subject sera. Suitable assays include, for example, ligand binding assays, such as radioimmunoassay (RIAs), ELISAs, and multiplex assays (Luminex, Bioplex, MSD); functional assays, such as opsonophagocytic assays (OPK, OPA), plaque reduction neutralization test (PRNT), or internalization assays; and in vivo assays in animal models of disease.

[0225] The RIA method detects specific antibodies through incubation of sera with radio-labeled polysaccharides or polypeptides in suspension (e.g., Schiffiman et al, 1980). The antigen-antibody complexes are then precipitated with ammonium sulfate and the radiolabeled pellets assayed for counts per minute (cpm).

[0226] In the ELISA detection method, specific antibodies from the sera of vaccinated subjects are quantitated by incubation with polysaccharides or polypeptides (either serotype-specific or conserved across two or more serotypes) which have been adsorbed to a solid support (e.g., Koskela and Leinonen (1981); Kojima et al, 1990; Concepcion and Frasch, 2001). The bound antibody is detected using enzyme-conjugated secondary detection antibodies. The ELISA also allows isotyping and subclassing of the immune response (i.e., IgM vs. IgG or IgGl vs. IgG2) by using isotype- or subclass-specific secondary antibodies and can be adapted to evaluate the avidity of the antibodies (Anttila et al, 1998; Romero-Steiner et al, 2005). Multiplex assays (e.g., Luminex) facilitate simultaneous detection of antibodies to multiple antigens. Capsular polysaccharide(s) or polypeptides are conjugated to spectrally distinct microspheres that are mixed and incubated with serum. The antibodies bound to the polysaccharides or polypeptides on the coated microspheres are detected using a secondary antibody (e.g., R-Phycoerythrin-conjugated goat anti-human IgG).

[0227] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by vaccines of the present invention. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which provides protection against pneumonia, mortality, or other endpoint is determined (Stack et al. 1998; Saeland et al. 2000).

[0228] In some embodiments, efficacy of vaccination may be determined by assaying one or more cytokine levels by stimulating T cells from a subject after vaccination. The one or more cytokine levels may be compared to the one or more cytokine levels in the same subject before vaccination. Increased levels of the one or more cytokine, such as a 1.5 -fold, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase over pre-immunization cytokine levels, would indicate an increased response to the vaccine. In some embodiments, the one or more cytokines are selected from GM-CSP; IL- la; IL- 1 P; IL- 2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F or other members of the IL-17 family; IL-22; IL-23; IFN-a; IFN- ; IFN-y; MIP-la; MIP-1P; TGF-P; TNFa, or TNF-p. In a nonlimiting example, efficacy of vaccination may be determined by assaying IL- 17 levels (particularly IL- 17A) by stimulating T cells from a subject after vaccination. The IL-17 levels may be compared to IL-17 levels in the same subject before vaccination. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold, 2- fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine.

[0229] In some embodiments, one may assay neutrophils in the presence of T cells or antibodies from the patient for viral killing. Increased viral killing, such as a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50- fold or 100-fold or more increase, would indicate an increased response to the vaccine. For example, one may measure TH 17 cell activation, where increased TH 17 cell activation, such as a 1.5 fold, 2-fold, 5- fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the vaccine. In another non-limiting example, one may measure THI cell activation, where increased THI cell activation, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the vaccine. One may also measure levels of an antibody specific to the vaccine, where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased vaccine efficacy. In certain embodiments, two or more of these assays are used. For example, one may measure IL- 17 levels and the levels of vaccine-specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of viral infection in vaccinated individuals compared to unvaccinated individuals.

[0230] Vaccine efficacy may also be assayed in various model systems such as the mouse challenge model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering the test vaccine to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of a pathogen. In some cases, a challenge dose administered intranasally or intratracheally is sufficient to cause pathogenic infection and/or a high rate of lethality in unvaccinated animals. One can then measure the reduction in infection and/or the reduction in lethality in vaccinated animals.

[0231] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by vaccines of the present invention. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which provides protection against bacteremia, colonization of organs or tissues, or mortality is determined (Stack et al. 1998; Saeland et al. 2000). Kits

[0232] The present disclosure also provides for kits for producing an immunogenic complex as disclosed herein which is useful for an investigator to tailor an immunogenic complex with their preferred polysaccharide and polypeptide antigens, e.g., for research purposes to assess the effect of an antigen, or a combination of antigens on immune response. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: a container comprising a polysaccharide cross-linked with a plurality of first affinity molecules; a container comprising a complementary affinity molecule which associates with the first affinity molecule, wherein the complementary affinity molecule associates with a polypeptide antigen, where the polypeptide antigen is from the same organism as the polysaccharide; a container comprising an antigen; a container comprising a fusion protein; a container comprising an antigen associated with a complementary affinity molecule; a container comprising a fusion protein associated with a complementary affinity molecule.

[0233] In another embodiment, the kit comprises a container comprising a polysaccharide; a container comprising a plurality of first affinity molecules; and a container comprising a cross-linking reagent for cross-linking the first affinity molecules to the polysaccharide, for example, but not limited to, CDAP (1- cyano-4- dimethylaminopyridinium tetrafluoroborate), and EDC (l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride).

[0234] In another embodiment, the kit comprises a container comprising a polypeptide antigen, and a container comprising a complementary affinity molecule, which associates with a first affinity molecule. In some embodiments, the kit further comprises a means to attach the complementary affinity molecule to the peptide antigen, where the means can be by a cross-linking reagent or by some intermediary protein.

[0235] In some embodiments, the kit can comprise at least one co-stimulation factor, which can be added to the polysaccharide or to another polymer. In some embodiments, the kit comprises a crosslinking reagent, for example, but not limited to, CDAP (l-cyano-4- dimethylaminopyridinium tetrafluoroborate); EDC (l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride); sodium cyanoborohydride; cyanogen bromide; and ammonium bicarbonate/iodoacetic acid, for linking the cofactor to the polysaccharide or to another polymer.

[0236] A variety of kits and components can be prepared for use in the methods described herein, depending upon the intended use of the kit, the particular target polysaccharide and polypeptide antigen and the needs of the user.

[0237] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0238] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

[0239] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1.A method of potentiating a B cell recall response to a polysaccharide antigen of a pathogen to a predetermined target level, the method comprising: administering to a subject an immunogenic composition comprising:

(a) a polysaccharide antigen of the pathogen; and

(b) at least one polypeptide antigen that is expressed by the pathogen; wherein the polysaccharide antigen is associated with the at least one polypeptide antigen; and wherein following administration of the immunogenic composition to the subject and subsequent exposure of the subject to the pathogen, the immunogenic composition potentiates a B cell recall response to the polysaccharide antigen to the predetermined target level.

2. The method of paragraph 1, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

(a) a biotinylated polysaccharide antigen comprising biotin and the polysaccharide antigen of the pathogen; and

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) the at least one polypeptide antigen that is expressed by the pathogen, wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotin-binding moiety of the fusion protein.

3. The method of paragraph 1, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen of the pathogen covalently conjugated to the at least one polypeptide antigen that is expressed by the pathogen.

4. The method of any one of paragraphs 1-3, wherein the predetermined target level is a level that is higher than the corresponding control level. 5. The method of paragraph 4, wherein the control level is a level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

6. The method of any one of paragraphs 1-5, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

7. The method of any one of paragraphs 1-6, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or by killing of the pathogen by immune sera from the subject in an opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

8. The method of any one of paragraphs 1-7, wherein the predetermined target level is determined based on a corresponding level of a B cell recall response induced in a non-human mammalian model upon administration of the immunogenic composition to the non-human mammalian model and subsequent exposure of the non-human mammalian model to the pathogen.

9. The method of any one of paragraphs 1-8, further comprising measuring the level of the B cell recall response in the subject following subsequent exposure to the pathogen.

10. The method of paragraph 9, wherein the measured level of the B cell recall response is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

11. The method of any one of paragraphs 1-10, further comprising confirming that the level of the B cell recall response after the subject has been exposed to the pathogen reaches the predetermined target level, e.g., a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

12. The method of any one of paragraphs 1-11, wherein the B cell recall response comprises activation and/or generation of memory B cells that are specific for the polysaccharide antigen.

13. The method of any one of paragraphs 1-12, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells.

14. The method of any one of paragraphs 1-13, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polypeptide antigen-specific Th cells. 15. The method of any one of paragraphs 1-14, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells and polypeptide antigen-specific Th cells.

16. A method of producing a B cell immune response to a polysaccharide antigen of a pathogen at a predetermined target level, the method comprising administering to a subject an immunogenic composition comprising an immunogenic complex, wherein the immunogenic complex comprises:

(a) a biotinylated polysaccharide antigen comprising biotin and a polysaccharide antigen of the pathogen; and

(b) a polypeptide comprising a biotin-binding moiety; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the polypeptide; and wherein the immunogenic complex, upon administration of the immunogenic composition to the subject, produces in the subject a B cell immune response to the polysaccharide antigen at a predetermined target level.

17. The method of paragraph 16, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen.

18. The method of paragraph 16 or 17, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at a level that is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen.

19. The method of any one of paragraphs 16-18, wherein the polypeptide comprising the biotin-binding moiety is a fusion protein comprising:

(i) the biotin-binding moiety; and

(ii) at least one polypeptide antigen.

20. The method of any one of paragraphs 16-19, wherein the B cell immune response is or comprises a MHC class II -dependent response.

21. A method of increasing uptake, processing, and/or presentation of a polysaccharide antigen from a pathogen by an antigen-presenting cell (APC) to a predetermined target level, the method comprising contacting an APC with an immunogenic composition comprising:

(a) a polysaccharide antigen of the pathogen; and

(b) a polypeptide; wherein the polysaccharide antigen is associated with the polypeptide; and wherein the immunogenic composition, upon contacting the APC, increases uptake, processing, and/or presentation of the polysaccharide antigen to the predetermined target level.

22. The method of paragraph 21, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

(b) the polypeptide, wherein the polypeptide comprises a biotin-binding moiety; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the polypeptide.

23. The method of paragraph 21, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen of the pathogen covalently conjugated to the polypeptide.

24. The method of any one of paragraphs 21-23, wherein the contacting comprises administering to a subject the immunogenic composition.

25. The method of any one of paragraphs 21-24, wherein the predetermined target level is determined based on a corresponding level of uptake, processing and/or presentation of the polysaccharide antigen upon contacting an APC in vitro with the immunogenic composition.

26. The method of any one of paragraphs 21-25, wherein the predetermined target level is at least 20% higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

27. The method of any one of paragraphs 21-26, wherein the predetermined target level is characterized by a level of intracellular polysaccharide antigen present in the APC being at least 5 -fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

28. The method of any one of paragraphs 21-27, wherein the predetermined target level is characterized by a level of polysaccharide antigen associated with the surface of the APC being at least 10-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with an antigenic polypeptide.

29. The method of any one of paragraphs 21-28, wherein the uptake, processing, and/or presentation of the polysaccharide antigen by the APC is or comprises a MHC class Il-dependent process.

30. A method of selecting an immunogenic composition candidate that induces immune responses to a polysaccharide antigen to a predetermined target level, the method comprising: contacting an antigen-presenting cell (APC) comprising MHC class II molecules with an immunogenic composition candidate, wherein the immunogenic composition candidate comprises:

(a) a polysaccharide antigen; and

(b) a polypeptide; wherein the polysaccharide antigen is associated with the polypeptide; characterizing uptake, processing, and/or presentation of the polysaccharide antigen on the MHC class II molecules by the APC, and selecting the immunogenic composition candidate as an agent useful for inducing immune responses to a polysaccharide antigen if the APC uptakes, processes, and/or presents the polysaccharide antigen on MHC class II molecules at a predetermined target level.

31. The method of paragraph 30, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

(a) a biotinylated polysaccharide antigen comprising biotin and the polysaccharide antigen; and

32. The method of paragraph 31, wherein the polypeptide comprising the biotin-binding moiety is a fusion protein comprising:

(i) the biotin-binding moiety; and

(ii) at least one polypeptide antigen.

33. The method of paragraph 30, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen covalently conjugated to the polypeptide.

34. The method of any one of paragraphs 30-33, wherein the predetermined target level is at least 2-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

35. The method of any one of paragraphs 30-34, wherein the predetermined target level is at least 5 -fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

36. The method of any one of paragraphs 30-35, wherein the predetermined target level is a level that is at least 10-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen. 37. The method of any one of paragraphs 30-36, wherein the characterizing comprises measuring a level of intracellular polysaccharide antigen present in the APC.

38. The method of any one of paragraphs 30-37, wherein the characterizing comprises measuring a level of polysaccharide antigen associated with the surface of the APC.

39. A method of immunizing a subject against a pathogen, the method comprising administering to a subject a dose of an immunogenic composition comprising an immunogenic complex, wherein the immunogenic complex comprises:

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) at least one polypeptide antigen that is expressed by the pathogen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein; and wherein the administered dose is lower than that of a reference composition to achieve in the subject, upon exposure to the pathogen, an equivalent or greater B cell recall response to the polysaccharide antigen.

40. The method of paragraph 39, wherein the B cell recall response of the immunized subject is characterized in that exposure of the immunized subject to the pathogen produces an antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at an equivalent or greater level to that produced by the reference composition.

41. A method of immunizing a subject against a pathogen, the method comprising administering to a subject a dose of an immunogenic composition comprising an immunogenic complex, wherein the immunogenic complex comprises:

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) at least one polypeptide antigen that is expressed by the pathogen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein; and wherein the administered dose provides protection against the pathogen for a longer period of time than provided by the same dose of a reference composition.

42. The method of any one of paragraphs 39-41, wherein the reference composition comprises a polysaccharide antigen that is not associated with a polypeptide antigen. 43. The method of any one of paragraphs 39-41, wherein the reference composition does not comprise a polypeptide antigen expressed by the pathogen.

44. The method of any one of paragraphs 39-41, wherein the reference composition comprises a polysaccharide antigen covalently conjugated to a carrier polypeptide.

45. The method of paragraph 41, wherein protection against the pathogen comprises a B cell recall response.

46. The method of paragraph 45, wherein the B cell recall response comprises an antibody response against the polysaccharide antigen induced by exposure of the subject to the pathogen, and wherein the immunogenic composition potentiates the B cell recall response to a level at least 20% higher than the corresponding level produced by administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

47. The method of paragraph 46, wherein the protection against the pathogen comprises a Thl response.

48. The method of paragraph 47, wherein the Thl response comprises production of IFN-y and/or TNF-a by CD4+ T cells at a level that is at least 1.1-fold higher than a corresponding level of IFN-y and/or TNF-a produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

49. The method of paragraph 46, wherein the protection against the pathogen comprises a Th 17 response.

50. The method of paragraph 49, wherein the Th 17 response comprises production of IL-17, IL-21, IL-22, IL24 and/or IL-26 by CD4+ T cells at a level that is at least 1. 1 -fold higher than a corresponding level of IL- 17, IL-21, IL-22, IL24 and/or IL-26 produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

51. The method of paragraph 46, wherein the protection against the pathogen comprises a CD8 response.

52. The method of paragraph 51, wherein the CD8 response comprises production of IFN-y, granzyme B, and/or perforin by CD8 T cells at a level that is at least 1.1-fold higher than a corresponding level of IFN-y, granzyme B, and/or perforin produced by CD8 T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

53. A method comprising: administering to a subject, who has received a prime MAPS vaccine against a pathogen, a booster vaccine comprising a polysaccharide antigen of the pathogen, wherein the MAPS vaccine comprises:

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) at least one polypeptide antigen that is expressed by the pathogen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein; and wherein the booster vaccine, upon administration to the subject that has received the prime MAPS vaccine, induces a B cell response to the polysaccharide antigen at a predetermined target level.

54. The method of paragraph 53, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine.

55. The method of paragraph 53 or 54, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine.

56. The method of any one of paragraphs 53-55, wherein the booster vaccine comprising the polysaccharide antigen is, or comprises, the MAPS vaccine, wherein the MAPS vaccine comprises:

(a) a biotinylated polysaccharide antigen comprising biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) at least one polypeptide antigen that is expressed by the pathogen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein

57. The method of any one of paragraphs 53-55, wherein the booster vaccine comprising the polysaccharide antigen is or comprises a preparation comprising a polysaccharide antigen that is not associated with a polypeptide antigen. 58. The method of any one of paragraphs 53-55, wherein the booster vaccine comprising the polysaccharide antigen is or comprises a polysaccharide antigen covalently conjugated to a carrier polypeptide.

59. The method of any one of paragraphs 53-58, comprising administering the MAPS vaccine to the subject prior to administering the booster vaccine.

60. A method comprising: administering to a subject, who has received a prime vaccine against a pathogen, a booster MAPS vaccine, wherein the MAPS vaccine comprises:

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

(ii) at least one polypeptide antigen that is expressed by the pathogen; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding moiety of the fusion protein; wherein the prime vaccine comprises a polysaccharide antigen of the pathogen; and wherein the booster MAPS vaccine, upon administration to the subject who has received the prime vaccine, induces a B cell response to the polysaccharide antigen at a predetermined target level.

61. The method of paragraph 60, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein.

62. The method of paragraph 60 or 61, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein.

63. The method of any one of paragraphs 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises the MAPS vaccine.

64. The method of any one of paragraphs 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises a preparation comprising a polysaccharide antigen that is not associated with a polypeptide antigen. 65. The method of any one of paragraphs 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises a polysaccharide antigen of covalently conjugated to a carrier polypeptide.

66. The method of any one of paragraphs 60-65, comprising administering the prime vaccine to the subject prior to administering the booster MAPS vaccine.

67. The method of any one of paragraphs 53-66, wherein the B cell response is a T helper (Th)-dependent response against the polysaccharide antigen and/or the polypeptide antigen.

68. The method of any one of paragraphs 1-67, wherein the pathogen is a Streptococcal (e.g., Group A, Group B, and Viridans), Staphylococcal (e.g., S. aureus), Meningococcal, Pneumococcal, Gram-Negative Bacteria (e.g., E. coli, Klebsiella, Pseudomonas, Enterobacter, Citrobacter, Acinetobacter, Serratia, Burkholderia, Salmonella, Shigella, and Bordetella), coronavirus, Mycobacterium (e.g., M. tuberculosis), Plasmodium (e.g., P. falciparum), pathogen.

69. The method of any one of paragraphs 1-68, wherein the immunogenic composition comprises a plurality of different species of immunogenic complexes, wherein the different species comprise different polysaccharide antigens, and/or different polypeptide antigens.

70. The method of any one of paragraphs 1-69, wherein the polysaccharide antigen is or comprises a portion of a capsular polysaccharide of Streptococcus pneumoniae.

71. The method of any one of paragraphs 1-70, wherein the capsular polysaccharide of Streptococcus pneumoniae is selected from: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 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.

72. The method of any one of paragraphs 1-71, wherein the polypeptide antigen is a polypeptide antigen selected from pneumococcal antigens (e.g., Group A, Group B, and Viridans antigens), tuberculosis antigens, anthrax antigens, HIV antigens, seasonal or epidemic flu antigens, Pertussis antigens, Staphylococcus aureus antigens, Meningococcal antigens, Haemophilus antigens, HPV antigens, Shigella antigens, Salmonella antigens, malaria antigens, Pseudomonas antigens, coronavirus antigens, or combinations thereof.

73. The method of any one of paragraphs 1-72, wherein the polypeptide antigen is an SP1500 polypeptide, an SP0785 polypeptide, and/or a pneumolysin polypeptide.

74. The method of any one of paragraphs 1-73, wherein the immunogenic composition is administered as part of a pharmaceutical composition further comprising a pharmaceutically acceptable earner. 75. The method of paragraph 74, wherein the pharmaceutical composition further comprises one or more adjuvants.

76. The method of paragraph 75, wherein the one or more adjuvants are or comprise a costimulation factor.

77. The method of paragraph 74 or 75, wherein the one or more adjuvants are selected from the group consisting of aluminum phosphate, aluminum hydroxide, and phosphated aluminum hydroxide.

78. The method of any one of paragraphs 75-77, wherein the one or more adjuvants are or comprise aluminum phosphate.

79. The method of any one of paragraphs 75-78, wherein the pharmaceutical composition is formulated for injection.

80. The method of any one of paragraphs 75-79, wherein the biotin-binding moiety is or comprises a dimeric biotin-binding moiety.

81. The method of any one of paragraphs 75-80, wherein the dimeric biotin-binding moiety is or comprises a rhizavidin polypeptide.

82. The method of any one of paragraphs 75-81, wherein the rhizavidin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or a biotin-binding fragment thereof.

83. The method of any one of paragraphs 1-82, wherein the polypeptide antigen is selected from any one or more of: a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12; a SP0785 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NOTO or a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NOT or SEQ ID NO: 4.

84. The method of any one of paragraphs 1-83, wherein the polypeptide antigen is a fusion protein, wherein the fusion protein is selected from any one or more of: a fusion protein, comprising, in any order, a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12, fused to a SP0785 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 10; a fusion protein, comprising, in any order, a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12, fused to a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NOT or SEQ ID NO: 4; or a fusion protein, comprising, in any order, a SP0785 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 10 fused to a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

85. The method of any one of paragraphs 1-84, wherein the polypeptide antigen, or fusion protein is further fused to a rhizavidin polypeptide having an amino acid sequence at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 2.

86. An immunogenic complex comprising:

(a) a biotinylated polysaccharide antigen comprising biotin and a polysaccharide antigen; and

(b) a biotin-binding polypeptide; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding polypeptide; and wherein the biotin-binding polypeptide does not include a polypeptide antigen from a pathogen.

87. An immunogenic complex comprising:

(b) a biotin-binding polypeptide; wherein the biotinylated polysaccharide antigen is non-covalently associated with the biotinbinding polypeptide; and wherein the biotin-binding polypeptide includes a polypeptide antigen from a pathogen.

88. The immunogenic complex of paragraph 86 or 87, wherein the biotin-binding polypeptide is or comprises avidin.

89. The immunogenic complex of paragraph 86 or 87, wherein the biotin-binding polypeptide is or comprises rhizavidin, wherein the rhizavidin comprises an amino acid sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 2.

90. The immunogenic complex of any of paragraphs 86-97, wherein the biotin-binding polypeptide further comprises a polypeptide antigen that is a tumor polyscaharride.

91. The immunogenic complex of paragraphs 86-90, wherein the capsular polysaccharide is Streptococcus pneumoniae polysaccharide selected from any of the: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, HD, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 4 IF, 42, 43, 44, 45, 46, 47A, 47F, and 48. The immunogenic complex of paragraphs 87-91, wherein the biotin-binding polypeptide further comprises a polypeptide antigen that is a coronavirus antigen. A vaccine composition, comprising two or more species of immunogenic complex according to paragraphs 86 or 87, wherein the combined weight of the polysaccharides (PS) and polypeptides in the vaccine contributed by each immunogenic complex is about the same , such that the w/w proteimPS ratio is about 1 : 1 or more than 1: 1. The vaccine composition of paragraph 93, wherein the w/w protein: PS ratio is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1: 10. The vaccine composition of paragraph 94, wherein the combined weight of the polysaccharide in the vaccine contributed by the immunogenic complexes ranges from about 0.2pg to about 12 pg. or 0.2 g to about 6 g, or 0.2 g to about 20 g, or 0.2 g to about 40 g. A vaccine composition, comprising two or more species of immunogenic complex according to paragraphs 86 or 87, wherein the combined weight of the polysaccharides (PS) and polypeptides in the vaccine contributed by each immunogenic composition is different, such that the w/w proteimPS ratio is not about 1: 1. The vaccine composition of paragraph 96, wherein the w/w protein: PS ratio is 2:l; 3: l, 4: l, 5: l, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1. The vaccine composition of paragraph 96, wherein the combined weight of the polysachharide and polypeptides in the vaccine contributed by each immunogenic complex ranges from about 0.4 pg to about HO g. The vaccine composition of any of paragraphs 93-98, comprising at least one immunogenic complex of paragraph 86 and at least one immunogenic complex of paragraph 87. . The vaccine composition of paragraph 96-99, comprising at least 15 or more species of an immunogenic composition according to paragraph 87. 1. The vaccine composition of any of paragraphs 93-100, wherein at least one immunogenic complex comprises a polysaccharide from any of Streptococcus pneumoniae serotypes selected from: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, HA, 11B, 11C, HD, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 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; and at least one immunogenic complex comprises a polysaccharide selected from any of: meningococcal polysaccharide, a Haemophilus influenze type b polysaccharide, a Streptococcus agalactiae polysaccharide, a Salmonella typhi Vi polysaccharide, a Klebsiella polysaccharide, a Pseudomonas polysaccharide, a Escherichia coli polysaccharide, or a Staphylococcus aureus polysaccharide, Hib (Haemophilus influenzae type B) capsular polysaccharide, meningococcal capsular polysaccharides, the polysaccharide of Bacillus anthracis (the causative agent of anthrax), Neisseria meningitidis (e.g., capsular polysaccharides selected from any of serogroups: A, C, W, W135, or Y). . The vaccine composition of any of paragraphs 93-101, wherein at least one immunogenic complex comprises a polysaccharide from Klebsiella pneumoniae, or a lipopolysaccharide (LPS)- derived polysaccharides, wherein the LPS-derived polysaccharides is a O polysaccharides (OPS). The vaccine composition of paragraph 102, wherein the LPS-derived polysaccharides is a core O polysaccharides (COPS), or is selected from, or derived from, an OPS from Klebsiella pneumoniae serotypes selected from: 01, 02, O2ac, 03, 04, 05, 07, 08, or 012, or a CPS from Klebsiella pneumoniae KI, K2, KI 0, KI 6, or KI 9. . The vaccine composition of any of paragraphs 93-103, wherein at least one immunogenic complex comprises a polysaccharide selected from: Type 5 or Type 8 polysaccharide, or any of the polysaccharides or oligosaccharides of Staphylococcus aureus. . The vaccine composition of any of paragraphs 93-104, wherein the vaccine is incorporated into any of: lysosome, cochleates, biodegradable polymers, or entrapped in microemulsions. . The vaccine composition of any of paragraphs 93-105, wherein the vaccine . The vaccine composition of any of paragraphs 93-105, wherein the composion further comprises one or more adjuvants, or a mixture of adjuvants. . The vaccine composition of paragraph 107, wherein the adjuvant is selected from at least one of: aluminium hydroxide, aluminium phosphate (Alum), cholera toxin, lalible toxids. . The vaccine composition of paragraph 107, further comprising a nucleic acid encoding a cytokine. . The vaccine composition of paragraphs 93-109, wherein the vaccine is formulated for administration or delivery to the mucosa. . The vaccine composition of any of paragraphs 93-110, wherein the vaccine composition is lyophilized. . The method of any of paragraphs 1-85, comprising administering a vaccine composition of any of paragraphs 93-111, or an immunogenic complex of any of paragraphs 86 or 87. . A fusion protein, comprising, in any order, a biotin-binding moiety comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO:2, and a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 4. . A MAPS vaccine for use in the methods of any of paragraphs 1-85, the MAPS vaccine comprising: a biotinylated polysaccharide antigen comprising biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein of paragraph 113.

115. The vaccine composition of any of paragraphs 93-111, comprising the MAPS vaccine of paragraph 114.

[0240] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.

EXEMPLIFICATION

Example 1. Immunization with MAPS vaccines induces T-dependent (TD) anti-CPS response and immune memory

[0241] C57BL/6mice were immunized with either type 14 pneumococcal CPS (CPS 14), or a MAPS vaccine consisting of biotinylated CPS14 coupled to avidin protein (i.e., CPS14 MAPS according to Table 1), or a conjugate vaccine (CV) consisting of CPS 14 conjugated to tetanus toxoid, all adjuvanted with aluminum phosphate (Alum), or Alum alone (negative control).

[0242] Table 1. MAPS complexes used in this study

[0243] Anti-CPS 14 IgM and IgG antibodies were measured two weeks after each immunization. Like human infants, adult mice do not respond well to pure CPS antigens: immunization with CPS 14 resulted in a low level of anti-CPS IgM but no IgG antibodies due to the T-independent (TI) activation of BCPS (Figure 1A, CPS 14). In contrast, immunization with either the MAPS vaccine or the conjugate vaccine induced high levels of IgM and IgG anti -PS antibodies (Figure 1A, MAPS), and was associated with antibody affinity-maturation (Figure IB). As shown in Fig 1C, MAPS-induced anti-CPS responses are MHCII -dependent, confirming the involvement of classical Th cells, rather than other types of T cells (e.g., NKT cells).

[0244] An important outcome of classical T-dependent (TD) immune responses is the generation of antigen-specific memory cells. The induction of CPS-specific memory cells by MAPS vaccine was evaluated using adoptive cell transfer experiments. Four groups of Rag F mice received adoptive transfer of splenocytes isolated from naive (SpN) or MAPS-immunized mice (SpM). One week later, Ragl^-/" mice received one immunization with either CPS 14 or CPS 14 MAPS. Anti-CPS IgM and IgG antibodies were measured two weeks after immunization. As shown in Figure ID, in Ragl ^/_ mice that received naive splenocytes, one immunization with CPS 14 (SpN, striped bars) or CPS 14 MAPS (SpN, open bars) induced typical primary responses, characterized by low concentrations of anti-CPS IgM and no detectable IgG antibodies. In contrast, in Ragl ^/_ mice that received splenocytes from MAPS- immunized mice, one immunization with CPS 14 MAPS led to increased anti-CPS IgM and, more importantly and surprisingly, robust production of anti-CPS IgG antibodies (Figure ID, SpM, open bars), reflecting recall responses in the presence of CPS-specific memory cells. Similar recall responses, associated with both anti-CPS IgM and IgG production, were also observed when SpM mice were immunized with CPS 14 alone (Figure ID, SpM, striped bars), albeit at lower levels than following immunization with MAPS. Thus, these results indicate that immunization with MAPS vaccine induces anti-CPS memory cells that can be re-activated (recalled) by either pure CPS (i.e., via a TI manner) or MAPS vaccines (i.e., via a TD manner), resulting in the production of a high level of anti-CPS IgG antibody. The difference between TI vs. TD recall responses is further examined in Example 2.

[0245] Figure 1. Panels A and B. C57BL/6mice (n=10 per group) received three subcutaneous immunizations with adjuvant alone (Alum), or adjuvanted CPS 14 or CPS 14 MAPS vaccine or CPS 14 conjugate vaccine (CV) (1 pg of CPS content per dose). Figure 1A. Anti-CPS IgM and IgG antibodies in each group after one (Pl), two (P2), or three immunizations (P3). Figure IB. Avidity of anti-CPS IgG antibodies in CPS14 MAPS-immunized mice aftertwo (P2) or three immunizations (P3). Figure 1C. Anti-CPS IgM and IgG antibodies in wild-type (WT) or MHCII ^/_ C57BL/6mice (n=5 per group) after one (Pl) or two (P2) immunizations with CPS 14 MAPS vaccine. Panel D. Ragl^-/" mice (n=8-10 per group) received an adoptive transfer of splenocytes isolated from naive mice (SpN) or splenocytes isolated from CPS 14 MAPS-immunized mice (SpM). Eight days post-adoptive transfer, Ragl ^/_ mice received one immunization with CPS 14 or CPS 14 MAPS (MAPS) (1 pg of PS content per dose). Anti- CPS IgG antibodies were measured 1 day prior (Pre) and 14 days after immunization (Post). For all panels, antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for CPS 14 antigen (see Materials and Methods). Bars represent geometric means + 95% CI. Statistical analyses were performed using the Mann-Whitney U test, in comparison to the Alum group (Figures 1A) or as indicated (Figures 1B-1D).

[0246] Materials and Methods

[0247] Mouse strains. Wild type, MHCII ^/_, and Ragl ^/_ C57BL/6mice (female, 5-6 weeks) were all purchased from Jackson Laboratories.

[0248] Ethics Statement. All procedures involving mice were approved by the Boston Children’s Hospital animal care and use committee (IACUC protocol number 19-10-4051R), following the National Institutes of Health guidelines for animal housing and care.

[0249] Cloning and protein purification. DNA sequences encoding SP1500 (amino acids 27-278) or SP0785 (amino acids 33-399) were amplified from .S', pneumoniae genomic DNA (purified from Tigr4 strain) via PCR. DNA sequence encoding pneumolysin toxoid (PdT) was obtained by PCR from pQE- 30-PdT plasmid (20). Purified PCR products were then cloned into a pET-21b vector for recombinant expression of C-terminal his6-tagged proteins. For rhizavidin fusion proteins, DNA sequences encoding indicated pneumococcal proteins were inserted at the 3 ’ end of the gene encoding the rhizavidin moiety in a pET-21b vector (20). All constructs were transformed into E. coli BL21 (DE3) strain for expression. His-tagged recombinant proteins were purified using Nitrilotriacetic acid (NTA) affinity chromatography (Qiagen) followed by size-exclusion chromatography using a Superdex 200 column (Cytiva). Protein concentration was determined using the BCA protein assay kit (Thermo scientific). Purified proteins were stored at -80 °C until use.

[0250] Preparation of MAPS Complexes. Avidin was purchased from Sigma Aldrich. Pneumococcal CPS were purchased from ATCC. Biotinylation of CPS was done as described previously using CDAP as the activation reagent (20). MAPS complexes were prepared by incubation of biotinylated CPS with indicated fusion proteins at room temperature overnight with an input ratio of protein to CPS at 3: 1 (w/w). Assembled MAPS complexes were purified by size -exclusion chromatography. CPS and protein concentration of purified MAPS complex was measured by anthrone assay (32) and BCA protein assay kit, respectively. Purified MAPS complex was stored at 4 °C in the presence of 0.01% thimerosal until use.

[0251] Immunizations and anti-sera production. All vaccines were prepared one day before immunization. Antigens were diluted to the appropriate concentration in saline, mixed with aluminum phosphate (Brenntag) (1.25 mg/mL final concentration of aluminum content), and then incubated at 4 °C overnight with rotation (24 rpm). Mice received subcutaneous immunization with the indicated vaccine in 200 pl, up to three times, two weeks apart. Sera were collected two weeks after each immunization or as indicated for antibody analysis.

[0252] For rabbit immune sera generation, New Zealand white rabbits (n=2 per group) received three intramuscular immunizations with CPS14-carrierl or CPS4-carrierl MAPS (1 pg of CPS content per dose), two weeks apart. Sera were collected two weeks after the last immunization and analyzed by ELISA against CPS 14 or CPS4. The serum that had the highest CPS-specific IgG antibody titer was used for inhibition ELISA.

[0253] Adoptive cell transfer. Donor mice were immunized once with indicated MAPS complex (at 1 pg of each CPS dosage) or fusion protein (10 pg) and then housed for at least 2 months before the collection of splenocytes. For splenocyte preparation, donor mice were euthanized and spleens were dissected and processed as described previously (33). B cells or CD4+ T cells were further purified from splenocytes using CD19 or CD4 MicroBeads (Miltenyi Biotec) according to the manufacture’s instruction. All cell preparations were then resuspended in PBS with 2% BSA. For adoptive transfer, each Ragl ^/- mouse received an intraperitoneal injection with 2.5 x lO⁷ of B cells, with or without an additional 1.25 x io⁷ of CD4⁺ T cells as indicated.

[0254] Statistical analysis. All statistical analyses were done using PRISM (version 5.01 for Windows, GraphPad Software, Inc). Differences between groups were compared using a nonparametric, two-tailed Mann-Whitney U test.

Example 2. Coupling CPS with fusion proteins enhances uptake, processing, and surface presentation of CPS antigens in APCs

[0255] Processing and presentation of CPS antigens, with or without coupling to fusion proteins, was examined. Peritoneal macrophages isolated from C57BL/6 mice were used for all assays. Binding and internalization of CPS antigens were examined after incubation with cells at 4 °C or 37 °C for various periods. The amount of CPS (in mg) present on the surface or inside the cells was measured by inhibition ELISA and then normalized to the total protein content of cell lysates (per mg). Uncoupled CPS 14 (non-biotinylated) had very little interaction with macrophages: there was minimum binding at 4 °C and barely detectable internalization (<0.1 mg) after overnight incubation at 37 °C (Figure 2A and 2B, CPS 14). In contrast, affinity-coupling to avidin (as a MAPS complex) greatly enhanced binding (-0.16 mg, Figure 2A, MAPS) and internalization of CPS14 in macrophages (Figure 2B). Prolonged incubation at 37 °C resulted in significant increases in both intracellular CPS (as a result of internalization) (Figure 2B, MAPS, grey bars) and surface-associated CPS (Figure 2B, MAPS, open bars). At the end of 6- or 18-h incubation, the amount of surface-associated CPS was 0.5 or 1.6 mg, respectively, which far exceeded the level of surface binding of CPS (in MAPS) measured at 4 °C or after 0.5 h-incubation at 37 °C (<0.2 mg). This increase may reflect the surface-presentation of CPS (epitopes) after intracellular processing of the internalized MAPS complexes.

[0256] To further characterize this surface -presentation of CPS, the interaction of CPS 14 MAPS with wild-type (WT) or MHCII^{- -} macrophages was examined. Also, in this experiment, heat-killed type 14 pneumococci (Pnl4) was included for comparison. While surrounded by many bacterial proteins, in Pnl4, the CPS is covalently attached to peptidoglycan and has no direct connection to proteins (Kolkman et al., Mol. Microbiol. 1997 26: 197-208). As expected, purified CPS14 (non-biotinylated) alone had minimal interaction with either type of macrophages (Figure 2C, CPS 14). In contrast, in the context of bacterial cells (proteins) a significant amount of Pnl4-CPS was captured by WT macrophages after overnight incubation. Over half of the CPS (~0.7 mg) accumulated inside the cells (Figure 2C, Pnl4, black bar), and the rest (-0.5 mg) was presented at the surface (after intracellular processing) (Figure 2C, Pnl4, white bar). The absence of MHCII molecules did not reduce surface -presentation of Pnl4-CPS (Figure 2C, Pnl4, striped bars), suggesting that this presentation is mediated via other molecules, possibly CDld (24, 25). In the case of CPS14 MAPS, overnight incubation led to -1.8 mg of total CPS associated with macrophages. Over 80% of these CPS (-1.5 mg) was localized on the cell surface (Figure 2C, MAPS, white bar), suggesting a more efficient processing/presentation ofMAPS-CPS compared to Pnl4-CPS. When incubated with MHCII^{- -} macrophages, the surface presentation of MAPS- CPS was reduced by - 40% compared to WT macrophages (Figure 2C, MAPS, white bar vs. white striped bar). The alternative, MHCII-independent presentation of MAPS-CPS in MHCII^7- macrophages was comparable to the presentation of Pnl4-CPS in either WT or MHCII-/- macrophages (Figure 2C, MAPS, striped white bar vs. Pnl4, white and striped white bars). The association of MAPS-CPS14 with MHCII molecules in WT macrophages was further confirmed by co-immunoprecipitation using anti- CPS14 antibodies followed by Western blot using anti-MHCII antibodies (Figure ID).

[0257] These results demontrate that either directly coupling CPS with fusion proteins (as in MAPS complexes) or indirectly associating CPS with proteins (as in bacterial cells) can significantly enhance uptake, intracellular processing, and presentation of CPS antigens in APCs. However, importantly, it was discovered that only in the case of MAPS complexes, where CPS is directly/tightly coupled to fusion proteins (i.e., a fusion protein comprising one or more polypeptide antigens), is the processed CPS presented via MHCII molecules.

[0258] Figure 2. Figures 2A and 2B. Peritoneal macrophages isolated from C57BL/6 mice were incubated in a culture medium containing no CPS (Con), 2.5 pg/mL of CPS 14, or CPS 14 MAPS (at 2.5 pg/mL of CPS content) at 4 °C for 2 hours (Figure 2A), or at 37 °C for indicated periods (Figure 2B). Intracellular and surface-associated CPS content in different samples were measured by inhibition ELISA and then normalized to the total cellular protein content (pg of CPS per mg of proteins). Bars represent means + SEM (n=12 in 4 independent experiments). Panel C. Internalization and presentation of purified CPS 14, or CPS 14 in bacterial cells or MAPS complex in wild-type (WT) or MHCII ^/- macrophages. Peritoneal macrophages isolated from either WT or MHCII^7- C57BL/6mice were incubated in a culture medium containing CPS 14, heat-killed type 14 pneumococci (Pnl4), or CPS 14 MAPS (all at 2.5 pg/mL of CPS content) at 37 °C for 18 hours. Intracellular and surface-associated CPS content in different samples were measured by inhibition ELISA and then normalized to the total cellular protein content. Bars represent means + SEM (n=9 in 3 independent experiments). Statistical analyses were performed using the Mann-Whitney U test between indicated groups. Panel D. Peritoneal macrophages were incubated with CPS 14 MAPS (2.5 pg/ml of CPS and 7.5 pg/ml of avidin), CPS 14 (2.5 pg/ml), or avidin (Avi) (7.5 pg/ml) at 37 °C for 18 hours. After incubation, cells were washed with PBS twice and then lysed with lysis buffer. All cell lysates were then normalized by total protein content measured by BCA assay. For co-immunoprecipitation, each cell lysate was mixed with rabbit anti-CPS14 serum pre-treated protein A resins and incubated overnight at 4 °C. After extensive washing with lysis buffer, the resins were boiled in SDS sample buffer and then the supernatants were applied onto SDS- PAGE. Western blot was done using primary antibodies against b-actin (internal control) and MHCII.

Example 3. Coupling with fusion proteins enables a dual-activation mechanism for naive BCPS mediated by TCPS and TFP

[0259] Presentation of antigens by MHCII leads to the activation of cognate Th cells. In the case of MAPS complexes, where CPS and fusion proteins comprising one or more polypeptide antigens are non- covalently coupled together, APCs can present the CPS (as shown in Example 2) and the fusion protein simultaneously (as indicated by the robust anti-fusion protein IgG production following vaccination with MAPS (Zhang et al., PNAS 2013 110: 13564-9 and Zhang et al. mBio. 2018 9) and Figure 7 in Example 6). This dual presentation demonstrates that two different populations of Th cells are activated: TCPS and Tpusion Protein (TFP). BCPS, as a special type of APCs, could then interact with TCPS or TFP, via MHCII- presented CPS or fusion protein, respectively.

[0260] The contribution of these two types of Th cells in the activation of naive BCPS during immunization with MAPS was investigated. Furthermore, the activation mechanism for different CPS antigens was also investigated.

[0261] The role of individual Th cell populations in the activation of naive BCPS was studied by adoptive transfer. As the frequency of TCPS or TFP is very low in naive mice, these Th cells were enriched by immunizing mice with one dose of MAPS vaccine or fusion protein. The role of TFPT was evaluated as follows: A 5-valent MAPS vaccine (5V-MAPS1) was made by coupling five biotinylated pneumococcal CPS, individually, with a fusion protein consisting of rhizavidin fused to two pneumococcal proteins SP1500 and SP0785 (fusion protein 1: CPI) (see Table 1 in Example 1). Two groups of donor mice were immunized with either 5V-MAPS1 (to enrich both TCPS and TFP) or just CPI (to enrich TFP). Six weeks later, B cells or CD4⁺ T cells from naive (TN), CPI-immunized (TC), or 5V-MAPS1 (TM) immunized mice were isolated and combinations of cells were adoptively transferred into Ragl ^/_ mice as shown in Figure 3. One week later, Ragl^-/" mice received one immunization with 5V-MAPS1. Preimmunization sera of Ragl ^/_ mice showed no detectable anti-CPS IgG antibodies (Figure 3, dashed line). Two weeks after vaccination, all Ragl ^/_ mice that received a combination of naive B cells (BN) with a source of CD4⁺ T cells, but not mice that received BN alone, developed anti-CPS IgG antibodies (Figure 3), reflecting TD activation of naive BCPS by MAPS vaccine. For all five CPS antigens, the group that received MAPS-primed Th cells (with enriched TCPS and TFP) had the most effective activation of BCPS and produced the highest amount of anti-CPS IgG (Figure 3, BN+TM). In contrast, transferring CPI- primed Th cells (with enriched TFPI) had a differential effect on the antibody response to various CPS antigens. For CPS5, CPS4 and CPS 14, there was a clear enhancement of anti-CPS IgG production in the presence of CPI-primed Th cells compared to naive Th cells; for CPS1 and CPS3, the difference between the two groups was less obvious (Figure 3, BN+TN vs BN+TC). This demonstrates that TFP can indeed mediate the activation of naive BCPS but that this mechanism is more relevant for some CPS antigens than others. Furthermore, the greater activation of BCPS to all five CPS antigens mediated by MAPS-primed Th cells suggests Tpp-independent activation mechanisms, likely mediated by TCPS-

[0262] To further evaluate the contribution of TCPS to BCPS activation, Ragl ^/_ mice received a transfer of naive B cells, alone or combined with naive, CPI-primed, or 5V-MAPS1 primed Th cells as in the first experiment, but were then immunized with 5V-MAPS2, a MAPS vaccine made with the same five CPS antigens but coupled to a different protein - egg avidin (not fused; Avi) (see Table 1 in Example 1). Avidin and CPI share no cross-reactive epitopes (Figures 8 and 9 in Example 6). Therefore, the presence of enriched TFPI to CPI should not provide additional help to B cell activation following immunization with 5V-MAPS2. Consistent with this prediction, following 5V-MAPS2 vaccination, there was no increase in anti -avidin or anti-CPS IgG production in Ragl ^/_ mice that received CPI -primed Th cells compared to that received naive Th cells (Figure 4, BN+TN vs. BN+TC, and Figure 9 in Example 6). In contrast, transferring 5V-MAPSl-primed Th cells enhanced IgG production for all five CPS antigens compared to naive or CPI Th cells (Figure 4, BN+TM vs. BN+TN or BN+TC). As the five CPS are the only antigens in common between 5V-MAPS1 and 5V-MAPS2, this enhanced anti-CPS IgG production is due to the contribution of TCPS (enriched in 5V-MAPS1 -primed Th cells) to BCPS activation. These results demonstrate that following immunization with MAPS, naive BCPS can be activated via two independent Th cell populations, TCPS or TFP, the relative contribution of which may depend on the individual CPS antigen.

[0263] Figure 3. Ragl ^/_ mice (n=5 for group BN and n=10 for other groups) received an adoptive transfer with B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), CPI -primed mice (TC), or 5V-MAPS1 -primed mice (TM). Eight days later, rag E ~ mice received one immunization with 5V-MAPS1 (1 mg per CPS). Anti-CPS IgG antibodies were measured 1 day prior (pre -immunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for each CPS antigen. Dashed lines indicate geometric means of anti-CPS IgG titers of all groups pre-immunization. Bars represent geometric means + 95% CI of anti-CPS IgG titer of each group post-immunization. Statistical analyses were performed using the Mann-Whitney U test between indicated groups.

[0264] Figure 4. Ragl ^/_ mice (n=5 for group BN and n=10 for other groups) received an adoptive transfer with B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), or CPI-primed mice (TC), or 5V-MAPS1 -primed mice (TM). Eight days after adoptive transfer, Ragl ^/_ mice received one immunization with 5V-MAPS2 (2.5 mg per CPS). Anti-CPS IgG antibodies were measured 1 day prior (pre-immunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for each CPS antigen. Dashed lines indicate geometric means of anti-CPS IgG titers of all groups pre-immunization. Bars represent geometric means + 95% CI of anti-CPS IgG titers of each group post-immunization. Statistical analyses were performed using the Mann-Whitney U test between indicated groups.

Example 4. Memory Th cells potentiate the recall anti-CPS responses mediated by memory BCPS

[0265] A direct outcome of TD activation of naive BCPS is the generation of memory BCPS which, upon re-exposure to CPS antigen, can rapidly upregulate IgG antibody production (a process referred to as a recall response). In contrast to primary responses, recall anti-CPS responses do not have to be induced by TD CPS antigens (e.g., CPS/protein conjugates or complexes). Clinically, recall anti-CPS responses have been seen when previously (conjugate vaccine) immunized individuals are boosted with pure CPS (e.g., Hib CPS or 23-valent pneumococcal CPS vaccine). The results shown in Fig, ID of Example 1 showed the same phenomenon in mice: a recall anti-CPS 14 response is observed following injection of pure CPS 14 in mice that received an adoptive transfer of MAPS-primed splenocytes. However, it is important to note that the CPS14-induced recall response is of a much lower magnitude than the recall response following exposure to CPS14 MAPS (a TD antigen) at the same dosage (1 mg of CPS).

[0266] To explore the differential anti-CPS recall response to TI vs. TD CPS antigens, the role of various Th cells in memory BCPS activation was examined. B cells from 5V-MAPS1 immunized mice (BM) were isolated as a source of memory BCPS • Isolated BM cells, alone or in combination with naive (TN), CPI -primed (TC), or 5V-MAPS1 -primed Th cells (TM) were adoptively transferred into Rag E mice. A separate group received naive B and Th cells (BN+TN) to provide a primary response baseline. One week after adoptive transfer, Ragl ^/_ mice received one immunization with 5V-MAPS1. No anti- CPS IgG was detected in pre-immunization sera (Figure 5, dashed lines). Two weeks after immunization, mice in all groups that received BM cells (containing memory BCPS), with or without Th cells, displayed robust recall responses, with anti-CPS IgG titers up to thousands fold higher than the primary response mediated by naive B and Th cells (Figure 5). In four out of five CPS antigens (except for CPS 14), the presence of naive Th cells provided no significant help to the activation of memory BCPS: the level of anti-CPS IgG production was comparable to the TI recall responses seen in the BM group (Figure 5, BM+TN vs. BM). In contrast, the presence of CPI-primed (with memory TFP) or MAPS-primed Th cells (with memory TCPS and TFP) significantly enhanced the recall anti-CPS IgG production by memory BCPS for these four CPS antigens (Figure 5, BM+TC or BM+TM vs. BM). In the case of CPS14, naive Th cells alone were sufficient to enhance recall responses. Together, this result confirms that memory BCPS can be activated in a TI manner and also demonstrates that a TD antigen (i.e., a MAPS complex) can induce enhanced recall anti-CPS responses, in most cases, via engaging memory (rather than naive) TFP and/or TCPS-

[0267] Figure 5. Ragl ^/_ mice (n=7-10 per group) received an adoptive transfer with B and CD4+ T cells isolated from naive mice (BN+TN); or with B cells isolated from 5V-MAPSl-primed mice (BM), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), CPI -primed mice (TC), or 5 V-MAPS1 -primed mice (TM). Eight days after adoptive transfer, Ragl ^/_ mice received one immunization with 5V-MAPS1 (1 mg per CPS content). Anti-CPS IgG antibodies were measured 1 day prior (pre-immunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for each CPS antigen. Dashed lines indicate geometric means of anti- CPS IgG titers of all groups pre-immunization. Bars represent geometric means + 95% CI of anti-CPS IgG titer of each group post-immunization. Statistical analyses were performed using the Mann-Whitney U test between indicated groups.

Example 5. Use of a pathogen-specific fusion protein enhances anti-CPS recall responses upon pathogen exposure

[0268] The results above revealed an important role of memory Th cells, especially memory TFP, in enhancing anti-CPS recall responses. Immunization with a CPS vaccine comprising a fusion protein (i.e., comprising a polypeptide antigen) derived from the target pathogen could induce memory TFP cells (that can be triggered during infections by the endogenous bacterial protein) was examined to determine if it could facilitate stronger anti-CPS recall responses.

[0269] A MAPS vaccine was prepared by coupling CPS4 with a fusion protein consisting of rhizavidin and PdT, a toxoid of pneumolysin (a pneumococcal cytolysin (Berry et al., Infect. Immun. 1999. 67:981-5), Ply) (see Table 1). Following one immunization with CPS4 MAPS, mice developed a strong antibody response to PdT and a low level of anti-CPS4 IgG (Fig 6 A and B, groups 3 and 4, Pre- SP exposure). Four weeks later, mice were intraperitoneally exposed to either heat-killed WT serotype 4 pneumococci or a heat-killed isogenic Ply knockout strain (dPly) (Figure 10 of Example 6); importantly, both killed bacterial preparations were normalized to have the same amount of CPS4 injected into mice. Surprisingly, when evaluated two weeks later, mice exposed to the killed WT strain produced a significantly higher amount of anti-CPS4 IgG than mice exposed to the killed dPly strain (Figure 6, groups 3 and 4, Post-SP exposure), confirming that prior exposure to the toxoid via immunization (to generate Ply-specific memory Th cells) would enhance the recall anti-CPS response to Ply-containing pneumococci. This demonstrates that a polypeptide antigen, when coupled to a CPS from the same pathogen, significantly potentiates the recall immune response against the pathogen.

[0270] Figure 6. C57BL/6 mice (n=10 for groups 1 and 2, n=15 for groups 3 and 4) received one immunization with adjuvant (Alum) alone (groups 1 and 2) or adjuvanted CPS4 MAPS vaccine (groups 3 and 4, 2 pg of CPS content per mouse). Serum samples were collected 14 days after immunization (Pre-SP exposure). Two weeks after the bleed, mice were exposed to either heat-killed wild type (WT) (groups 1 and 3) or pneumolysin knockout Tigr4 strain (dPly) (groups 2 and 4), each at 1 pg of CPS4 content (2.5 or 2.1 pg of protein content for WT or dPly, respectively) per mouse via intraperitoneal injection. Serum samples were collected 14 days after exposure (Post-SP exposure). Anti-Ply (Panel A) and anti-CPS IgG antibodies (Panel B) were measured using ELISA. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum of Ply or CPS4 antigen. Bars represent geometric means + 95% CI. Statistical analyses were performed using the Mann-Whitney U test between indicated groups.

Example 6. Additional studies

[0271] Figure 7. Immunization of mice with carrierl protein or 5V-MAPS1 generates robust anti-carrierl antibody responses. C57BL/6 mice (n=10 per group) received no immunization (Naive), or one immunization with CP 1 or 5 V-MAPS 1 , each at 15 pg of CP 1 content per mouse . Anti-CP 1 IgG antibodies were measured 14 days later. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for CPI. Bars represent geometric means + 95% CI. Statistical analyses were performed using the Mann-Whitney U test compared to the Naive group or between carrierl and 5V-MAPS 1 groups.

[0272] Figure 8. Rhavi- specific Th2 cells do not cross-react with egg avidin. C57BL/6 mice received either no immunization (Naive, n=5), or three immunizations with rhavi (Rhavi, n=10) at 5 pg per mouse. Antigen-specific Th2 responses were examined by IL-5 production after ex vivo stimulation of peripheral blood samples of immunized mice with rhavi or egg avidin (both at 10 mg/ml in DMEM/F12 medium containing 10% low -endotoxin defined FBS (Hyclone), 50pM 2-mercaptoethanol (Sigma) and ciprofloxacin (10 pg/mL, Cellgro). After incubation at 37 °C for six days, culture supernatants were collected and the concentration of IL-5 in each samples was measured using IL-5 ELISA kit (Biolegend). Rhavi, but no egg avidin, activated Th2 cells of rhavi-immunized mice, leading to IL-5 production.

[0273] Figure 9. The presence of carrierl-primed or 5V-MAPSl-primed Th cells does not enhance anti-carrier2 IgG production during 5V-MAPS2 vaccination. Ragl ^/_ mice (n=I0 per group) received an adoptive transfer with B cells purified from naive mice (BN), alone or in combination with CD4⁺ T cells purified from naive mice (TN), or CPI -primed mice (TC), or 5V-MAPS1 -primed mice (TM) as described in Figure 4. Eight days after adoptive transfer, Ragl ^/_ mice received one immunization with 5V-MAPS2. Anti-CPl and anti-avidin IgG antibodies were measured 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum for CPI or avidin. Bars represent geometric means + 95% CL Statistical analyses were performed using the Mann-Whitney U test; n.s, not significant.

[0274] Figure 10. Western blot of heat-killed wild-type or pneumolysin knockout Tigr4 strain. Heat-killed wild type (WT) or pneumolysin knockout (dPly) Tigr4 pneumococci were loaded on SDS- PAGE (~30 pg of total protein content per sample) and then transferred to PVDF membrane for Western blot using rabbit immune sera against pneumolysin (Ply), or SP0785 or SP1500 (as positive controls). Compared to the WT strain, the dPly strain showed no Ply expression, but normal expression of SP0785 or SP1500.

[0275] Figure 11. The avidin protein in MAPS complex is more resistant to proteinase digestion and remains bound to biotinylated CPS even after being partially digested. MAPS complex was made with biotinylated CPS 14 and avidin. For in vitro digestion, each sample contains 2 pg of avidin protein in the form of MAPS complex or as free protein. Samples were incubated with or without proteinase K (0.5 mg/ml) at 37 °C for 8 hours and then treated with reduced SDS sample buffer at room temperature or boiling for 10 min before applied onto SDS-PAGE gel. For MAPS samples, the avidinbiotin interaction is stable in the presence of SDS at room temperature, and thus avidin protein remains in the loading well of the gel due to the big size of biotinylated CPS 14 (line 1, red arrow). After boiling, the avidin protein is dissociated from biotinylated CPS14 and runs into the gel (line 2). For free protein samples, avidin runs into the gel with or without boiling (line 5 and 6). After protease K treatment, about 73% (the intensity of Avi band in line 4 compared to Avi band in line 2) of the avidin proteins in MAPS complex remain intact (line 4, Avi) and the rest were partially digested (line 4, Avi F). Nevertheless, the partially digested avidin can still bind onto biotinylated CPS 14 if the sample was kept at room temperature (line 3, red arrow). In contrast, after the same protease K treatment, only 6% (the intensity of Avi band in line 8 compared to Avi band in line 6) of biotin-free avidin was left undigested (line 8, Avi). [0276] Materials and Methods

[0277] Binding, internalization, and presentation of CPS antigens in macrophages.

[0278] Peritoneal macrophages were isolated from C57BL/6 mice after intraperitoneal injection with 3% Brewer thioglycollate medium (3 mL per mouse) and stimulation for 3 days. To collect the macrophages, mice were euthanized, the abdominal skin was retracted, and 10 mL of cold PBS was injected along the left side of the peritoneal wall. The cell suspension was then aspirated using a syringe and dispensed into a 50-mL conical tube on ice. Cells were pelleted by centrifugation, resuspended in culture medium (DMEM/F12 medium with 10% FBS) and plated in 12-well plates at lx 10⁶ cells per mL per well. Unattached cells were removed after 3-hour incubation at 37 °C, and 0.5 mL of fresh medium containing plain CPS 14, MAPS complex or heat-killed bacteria (all at 2.5 pg/mL of CPS content) was added to each well for incubation at 4 °C or 37 °C for the indicated time (4 wells per condition). After incubation, the medium was discarded, and cells were washed with PBS twice before further treatment for CPS analysis.

[0279] For CPS quantification, we measured total cell-associated CPS and intracellular CPS content at each condition by inhibition ELISA and then calculated surface-associated CPS based on the difference between these two measurements. For every 6 wells of cells treated at the same condition, 3 wells were incubated with 1 :200 dilution of pre-immune rabbit sera (for measurement of the total cell-associated CPS) and 3 wells were incubated with 1:200 dilution of rabbit anti-CPS14 sera (to block surface- associated CPS for measurement of the intracellular CPS content) for 30 minutes RT. After incubation, cells were washed PBS three times and then incubated with cold water containing 2 mM EDTA and proteinase inhibitor cocktail (Thermo Scientific) (150 pL per well) at 4 °C for 1 hour. Cells were then detached from the plates by pipetting, and then transferred into PCR tubes and lysed by sonication in an ice-water bath. At the end of sonication, 15 pL of 10x PBS concentrate was added into each sample to adjust the pH to 7.5. CPS content in each sample was measured by inhibition ELISA and then normalized to the concentration of total cellular proteins measured by the BCA protein assay kit.

[0280] For Western blot and co-immunoprecipitation, 5x 10⁶ peritoneal macrophages were seeded in a 60 mm dish with 5 mL culture medium. Unattached cells were removed after 3-hour incubation at 37 °C, and 3 mL of fresh medium containing 2.5 mg/mL of CPS 14 or MAPS complex (in CPS content), or 7.5 mg/mL of avidin protein was added. After 18-hour incubation at 37 °C, the medium was discarded, and cells were washed with PBS twice, detached from the dish and lysed in 250 mL of cold lysis buffer (20 mM Tris, pH7.5, 150 mM NaCl, 2mM EDTA, 0.1% Triton X-100 and proteinase inhibitor cocktail) by sonication. For co-immunoprecipitation, cell lysates were mixed with protein A resin (GE Healthcare Life Science) that had been pre-treated with rabbit anti-CPS14 serum and then washed extensively with PBS for overnight incubation at 4 °C. After incubation, the resin was pelleted, washed with lysis buffer extensively and then boiled in 30 ml of reduced SDS sample buffer. Cell lysates and co- immunoprecipitate samples were applied to SDS-PAGE and then transferred onto PVDF membrane for Western blot using rabbit anti-avidin sera, rabbit monoclonal antibody against b-actin (Abeam) and rabbit polyclonal antibody against MHC-II (Abeam), as primary antibodies followed by HRP -conjugated anti-rabbit secondary antibody. The membrane was developed using ECL substrate (Thermo Scientific) and exposed on film.

[0281] Preparation of heat-killed pneumococci. Pneumococcal strain 1401 (serotype 14) and Tigr4 (serotype 4) were used in this study. A pneumolysin knockout Tigr4 strain was constructed using the procedure described previously (1, 2). Bacteria were grown in Todd-Hewitt medium plus 0.5% yeast extract (THY) at 37°C with 5% CO2 to ODgoo=0.05, and the transformation was done as described previously (3). Transformants were selected on Trypticase soy agar plates with 5% sheep blood (BAP), supplemented with 400 pg/mL Kanamycin and 600 pg/mL gentamicin. Mutants were confirmed by PCR and sequencing the genomic region of pneumolysin, as well as by Western blot probing for the pneumolysin protein.

[0282] To prepare heat-killed pneumococci, pneumococcal strains were streaked onto BAP and grown overnight at 37°C with 5% CO2. The overnight culture was collected in the morning and inoculated into THY medium and grown at 37°C with 5% CO2 until ODgoo=0.8. Bacteria were then pelleted by centrifugation, washed twice with PBS, resuspended in PBS (to ODgoo=4) and heat-inactivated by incubation at 58°C for 1 hour. After inactivation, a sample of bacteria was plated on BAP and cultured overnight at 37°C with 5% CO2 for confirmation of a complete killing. Protein and CPS concentrations of heat-killed pneumococci were determined by BCA assay and inhibition ELISA, respectively.

[0283] Antibody analysis. Antigen-specific antibodies were measured by ELISA. For CPS antigens, Immulon 2 HB 96-microwell plates (Thermo Scientific) were coated with CPS solution (0.5-5 pg/mL in PBS) for 5 hours at 37 °C and then overnight at 4 °C. For proteins, plates were coated with indicated protein (1 pg/mL in PBS) overnight at room temperature. Coated plates were washed with PBS containing 0.05% Tween 20 (PBST) and then blocked with 1% BSA in PBS for 1 hour. After blocking, serial dilutions of the reference serum and sample sera were added and incubated for 2 hours, followed by a 1-hour incubation with HRP-conjugated secondary antibody against mouse IgM or IgG. The plates were then washed and developed with SureBlue TMB Microwell Peroxidase Substrate (KPL). IM HC1 was used to terminate the reactions before the A45011111 was analyzed using an ELISA reader. Antibody titers were expressed in arbitrary units relative to the reference serum (generated by pooling sera from mice that have been immunized three times with 5V MAPS1 or with indicated carrier proteins). Antigenspecific IgM or IgG titer of each reference serum was arbitrarily assigned as 12,000 units/mL. [0284] IgG avidity was measured using the method described previously (4). Briefly, serum samples were diluted with PBST to the appropriate concentration and then added to CPS14-coated microplates (8 wells per sample, 100 pL per well) for a 2-hour incubation. After washing with PBST, seven 2-fold serial dilutions of sodium thiocyanate (NaSCN) (4M, in H2O) or a blank (H2O only) were added to the wells of each sample (100 pL per well) and incubated for 30 min. Plates were then washed, incubated with HRP- conjugated secondary antibody for 1 hour, and washed again before development with SureBlue. Avidity was expressed as the avidity index (A.I.), the molar concentration of NaSCN that elutes 50% of CPS14- specific IgG antibodies that bind onto the plates.

[0285] Inhibition ELISA for CPS quantification. CPS content in heat-killed pneumococci or different cellular preparations was measured by inhibition ELISA using polyclonal rabbit sera against CPS14 or CPS4. 96-well microplate was coated with CPS14 or CPS4 and then blocked with 1% BSA in PBS. In a separate assay microplate (not treated for ELISA antigen coating), in each well, 50 pL of samples or purified CPS (at different concentrations, as standard reference) were mixed with 50 pL of rabbit anti-CPS14 sera (1: 1600 dilution) or anti-CPS4 sera (1:800 dilution), incubated for 30 min, and then the mixture was transferred into the ELISA plate for an additional incubation of 2 hours. The plate was then washed, incubated with an HRP -conjugated secondary antibody and developed as described above for regular ELISA. The concentration of CPS in each sample was calculated according to the standard reference.

[0286] References Hua CZ, Howard A, Malley R, Lu YJ. 2014. Effect of nonheme iron-containing ferritin Dpr in the stress response and virulence of pneumococci. Infect Immun 82:3939-47. Sung CK, Li H, Claverys JP, Morrison DA. 2001. An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67:5190-6. Bricker AL, Camilli A. 1999. Transformation of a type 4 encapsulated strain of Streptococcus pneumoniae. FEMS Microbiol Lett 172: 131-5. Colino J, Duke L, Snapper CM. 2013. Noncovalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact grampositive extracellular bacteria. J Immunol 191:3254-63.

Equivalents

[0287] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

LIST OF SEQUENCES

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

MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALAFDASNFKDFSSIASASSSWQN QSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATG WTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD

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

FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGT FIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTT ENKSLLKD

SEQ ID NO: 3, Streptococcus pneumoniae Pneumolysin protein:

MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTAT NDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVND LLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQI VNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKS DEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRF TADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELS

YDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDL PLVRKRTISIWGTTLYPQVEDKVEND

SEQ ID NO: 4, Streptococcus pneumoniae Pneumolysin protein with mutations D385N, C428G, and W433F, denoted PdT:

YNHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREGTGLAFEWWRTVYEKTDLP LVRKRTISIWGTTLYPQVEDKVEND

SEQ ID NO: 9, SP0785 protein, full-length [aa 1-399], TIGR4 strain: (Note: One T394A mismatch with SP0785 NCBI Sequences ABJ54007.1 and YP816180) MKKKNGKAKKWQLYAAIGAASVVVLGAGGILLFRQPSQTALKDEPTHLVVAKEGSVASSVLLS GTVTAKNEQYVYFDASKGDLDEILVSVGDKVSEGQALVKYSSSEAQAAYDSASRAVARADRHI NELNQARNEAASAPAPQLPAPVGGEDATVQSPTPVAGNSVASIDAQLGDARDARADAAAQLSK AQSQLDATTVLSTLEGTVVEVNSNVSKSPTGASQVMVHIVSNENLQVKGELSEYNLANLSVGQ EVSFTSKVYPDKKWTGKLSYISDYPKNNGEAASPAAGNNTGSKYPYTIDVTGEVGDLKQGFSV NIEVKSKTKAILVPVSSLVMDDSKNYVWIVDEQQKAKKVEVSLGNADAENQEITSGLTNGAKVI

SNPTSSLEEGKEVKADEATN

SEQ ID NO: 10, SP0785 protein lacking signal sequence [aa 33-399]: (Note: One T394A mismatch with SP0785 NCBI Sequences ABJ54007.1 and YP816180)

FRQPSQTALKDEPTHLVVAKEGSVASSVLLSGTVTAKNEQYVYFDASKGDLDEILVSVGDKVSE GQALVKYSSSEAQAAYDSASRAVARADRHINELNQARNEAASAPAPQLPAPVGGEDATVQSPT PVAGNSVASIDAQLGDARDARADAAAQLSKAQSQLDATTVLSTLEGTVVEVNSNVSKSPTGAS QVMVHIVSNENLQVKGELSEYNLANLSVGQEVSFTSKVYPDKKWTGKLSYISDYPKNNGEAAS PAAGNNTGSKYPYTIDVTGEVGDLKQGFSVNIEVKSKTKAILVPVSSLVMDDSKNYVWIVDEQ QKAKKVEVSLGNADAENQEITSGLTNGAKVISNPTSSLEEGKEVKADEATN

SEQ ID NO: 11, SP1500 protein, full-length [aa 1-278], TIGR4 strain:

MKKWMLVLVSLMTALFLVACGKNSSETSGDNWSKYQSNKSITIGFDSTFVPMGFAQKDGSYA GFDIDLATAVFEKYGITVNWQPIDWDLKEAELTKGTIDLIWNGYSATDERREKVAFSNSYMKNE QVLVTKKSSGITTAKDMTGKTLGAQAGSSGYADFEANPEILKNIVANKEANQYQTFNEALIDLK NDRIDGLLIDRVYANYYLEAEGVLNDYNVFTVGLETEAFAVGARKEDTNLVKKINEAFSSLYK DGKFQEISQKWFGEDVATKEVKEGQ

SEQ ID NO: 12, SP1500 [aa 27-278]:

TSGDNWSKYQSNKSITIGFDSTFVPMGFAQKDGSYAGFDIDLATAVFEKYGITVNWQPIDWDLK EAELTKGTIDLIWNGYSATDERREKVAFSNSYMKNEQVLVTKKSSGITTAKDMTGKTLGAQAG SSGYADFEANPEILKNIVANKEANQYQTFNEALIDLKNDRIDGLLIDRVYANYYLEAEGVLNDY NVFTVGLETEAFAVGARKEDTNLVKKINEAFSSLYKDGKFQEISQKWFGEDVATKEVKEGQ

SEQ ID NO: 13, Ply gene encoding Ply protein, full-length [aa 1-470]

ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCT TGACCCATCAGGGAGAAAGTATTGAAAATCGTTTCATCAAAGAGGGTAATCAGCTACCCGA TGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGATATTTCT

GTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTT GTTAGAGAATAATCCCACTCTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTT GCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTC GCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGT

CCCAGCTAGAATGCAGCATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAA

GTTTGGTTCTGACTTTGAAAAGATAGGGAATTCTCTTGATATTGATTTTAACTCTGTCCATTC

AGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGAT

GCTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAGGCAGA

GAGGAATTTCTGCAGAGCGTCCTTTGGTCTATATTTCGAGTGTTGCTTATGGGCGCCAAGTC

TATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAATCTTTGAT

AAAAGGAGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGC

GGTTATTTTAGGGGGCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATG

GTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCCGATCATCCAGGCTTGCCGATTTC

CTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATG

TTGAGACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTAT

GTTGCTCAATATTATATTACTTGGGATGAATTATCCTATGATCATCAAGGCAAGGAAGTCTT

GACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAGTATTC

CTTTAAAAGGGAATGTTCGCAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGG

GAATGGTGGCGTACGGTTTATGAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTT

CTATTTGGGGAACAACTCTCTATCCTCAGGTAGAGGATAAGGTAGAAAATGATTAG

SEQ ID NO: 17, SP0785 gene encoding SP0785 protein, full-length [aa 1-399], TIGR4 strain:

ATGAAGAAAAAGAATGGTAAAGCTAAAAAGTGGCAACTGTATGCAGCAATCGGTGCTGCG

AGTGTAGTTGTATTGGGTGCTGGGGGGATTTTACTCTTTAGACAACCTTCTCAGACTGCTCT

AAAAGATGAGCCTACTCATCTTGTTGTTGCCAAGGAAGGAAGCGTGGCCTCCTCTGTTTTAT

TGTCAGGGACAGTAACAGCAAAAAATGAACAATATGTTTATTTTGATGCTAGTAAGGGTGA

TTTAGATGAAATCCTTGTTTCTGTGGGCGATAAGGTCAGCGAAGGGCAGGCTTTAGTCAAGT

ACAGTAGTTCAGAAGCGCAGGCGGCCTATGATTCAGCTAGTCGAGCAGTAGCTAGGGCAGA

TCGTCATATCAATGAACTCAATCAAGCACGAAATGAAGCCGCTTCAGCTCCGGCTCCACAG

TTACCAGCGCCAGTAGGAGGAGAAGATGCAACGGTGCAAAGCCCAACTCCAGTGGCTGGA

AATTCTGTTGCTTCTATTGACGCTCAATTGGGTGATGCCCGTGATGCGCGTGCAGATGCTGC

GGCGCAATTAAGCAAGGCTCAAAGTCAATTGGATGCAACAACTGTTCTCAGTACCCTAGAG

GGAACTGTGGTCGAAGTCAATAGCAATGTTTCTAAATCTCCAACAGGGGCGAGTCAAGTTA

TGGTTCATATTGTCAGCAATGAAAATTTACAAGTCAAGGGAGAATTGTCTGAGTACAATCTA

GCCAACCTTTCTGTAGGTCAAGAAGTAAGCTTTACTTCTAAAGTGTATCCTGATAAAAAATG

GACTGGGAAATTAAGCTATATTTCTGACTATCCTAAAAACAATGGTGAAGCAGCTAGTCCA

GCAGCCGGGAATAATACAGGTTCTAAATACCCTTATACTATTGATGTGACAGGCGAGGTTG

GTGATTTGAAACAAGGTTTTTCTGTCAACATTGAGGTTAAAAGCAAAACTAAGGCTATTCTT

GTTCCTGTTAGCAGTCTAGTAATGGATGATAGTAAAAATTATGTCTGGATTGTGGATGAACA ACAAAAGGCTAAAAAAGTTGAGGTTTCATTGGGAAATGCTGACGCAGAAAATCAAGAAAT

CACTTCTGGTTTAACGAACGGTGCTAAGGTCATCAGTAATCCAACATCTTCCTTGGAAGAAG

GAAAAGAGGTGAAGGCTGATGAAGCAACTAAT

SEQ ID NO: 18, SP1500 gene encoding SP1500 protein, full-length [aa 1-278], TIGR4 strain:

ATGAAAAAATGGATGCTTGTATTAGTCAGTCTGATGACTGCTTTGTTCTTAGTAGCTTGTGG

GAAAAATTCTAGCGAAACTAGTGGAGATAATTGGTCAAAGTACCAGTCTAACAAGTCTATT

ACTATTGGATTTGATAGTACTTTTGTTCCAATGGGATTTGCTCAGAAAGATGGTTCTTATGC

AGGATTTGATATTGATTTAGCTACAGCTGTTTTTGAAAAATACGGAATCACGGTAAATTGGC

AACCGATTGATTGGGATTTGAAAGAAGCTGAATTGACAAAAGGAACGATTGATCTGATTTG

GAATGGCTATTCCGCTACAGACGAACGCCGTGAAAAGGTGGCTTTCAGTAACTCATATATG

AAGAATGAGCAGGTATTGGTTACGAAGAAATCATCTGGTATCACGACTGCAAAGGATATGA

CTGGAAAGACATTAGGAGCTCAAGCTGGTTCATCTGGTTATGCGGACTTTGAAGCAAATCC

AGAAATTTTGAAGAATATTGTCGCTAATAAGGAAGCGAATCAATACCAAACCTTTAATGAA

GCCTTGATTGATTTGAAAAACGATCGAATTGATGGTCTATTGATTGACCGTGTCTATGCAAA

CTATTATTTAGAAGCAGAAGGTGTTTTAAACGATTATAATGTCTTTACAGTTGGACTAGAAA

CAGAAGCTTTTGCGGTTGGAGCCCGTAAGGAAGATACAAACTTGGTTAAGAAGATAAATGA

AGCTTTTTCTAGTCTTTACAAGGACGGCAAGTTCCAAGAAATCAGCCAAAAATGGTTTGGA

GAAGATGTAGCAACCAAAGAAGTAAAAGAAGGACAG

Claims

1. A method of potentiating a B cell recall response to a polysaccharide antigen of a pathogen to a predetermined target level, the method comprising: administering to a subject an immunogenic composition comprising:

(a) a polysaccharide antigen of the pathogen; and

2. The method of claim 1, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

3. The method of claim 1, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen of the pathogen covalently conjugated to the at least one polypeptide antigen that is expressed by the pathogen.

4. The method of any one of claims 1-3, wherein the predetermined target level is a level that is higher than the corresponding control level.

5. The method of claim 4, wherein the control level is a level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

6. The method of any one of claims 1-5, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

7. The method of any one of claims 1-6, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or by killing of the pathogen by immune sera from the subject in an opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

8. The method of any one of claims 1-7, wherein the predetermined target level is determined based on a corresponding level of a B cell recall response induced in a non-human mammalian model upon administration of the immunogenic composition to the non-human mammalian model and subsequent exposure of the non-human mammalian model to the pathogen.

9. The method of any one of claims 1-8, further comprising measuring the level of the B cell recall response in the subject following subsequent exposure to the pathogen.

10. The method of claim 9, wherein the measured level of the B cell recall response is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

11. The method of any one of claims 1-10, further comprising confirming that the level of the B cell recall response after the subject has been exposed to the pathogen reaches the pre -determined target level, e.g., a level that is at least 20% higher than the corresponding level of a B cell recall response induced in a subject following administration of an immunogenic composition that does not comprise a polypeptide antigen expressed by the pathogen.

12. The method of any one of claims 1-11, wherein the B cell recall response comprises activation and/or generation of memory B cells that are specific for the polysaccharide antigen.

13. The method of any one of claims 1-12, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells.

14. The method of any one of claims 1-13, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polypeptide antigen-specific Th cells.

15. The method of any one of claims 1-14, wherein the B cell recall response comprises activation of polysaccharide antigen-specific B cells via interaction with polysaccharide antigen-specific T helper (Th) cells and polypeptide antigen-specific Th cells.

17. The method of claim 16, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen.

18. The method of claim 16 or 17, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at a level that is at least 20% higher than the corresponding level of a B cell immune response produced in a subject by administration of an immunogenic composition comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen.

19. The method of any one of claims 16-18, wherein the polypeptide comprising the biotinbinding moiety is a fusion protein comprising:

(i) the biotin-binding moiety; and

(ii) at least one polypeptide antigen.

20. The method of any one of claims 16-19, wherein the B cell immune response is or comprises a MHC class II -dependent response.

(a) a polysaccharide antigen of the pathogen; and

22. The method of claim 21, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

23. The method of claim 21, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen of the pathogen covalently conjugated to the polypeptide.

24. The method of any one of claims 21-23, wherein the contacting comprises administering to a subject the immunogenic composition.

25. The method of any one of claims 21-24, wherein the predetermined target level is determined based on a corresponding level of uptake, processing and/or presentation of the polysaccharide antigen upon contacting an APC in vitro with the immunogenic composition.

26. The method of any one of claims 21-25, wherein the predetermined target level is at least 20% higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

27. The method of any one of claims 21-26, wherein the predetermined target level is characterized by a level of intracellular polysaccharide antigen present in the APC being at least 5 -fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

28. The method of any one of claims 21-27, wherein the predetermined target level is characterized by a level of polysaccharide antigen associated with the surface of the APC being at least 10-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with an antigenic polypeptide.

29. The method of any one of claims 21-28, wherein the uptake, processing, and/or presentation of the polysaccharide antigen by the APC is or comprises a MHC class Il-dependent process.

(a) a polysaccharide antigen; and

31. The method of claim 30, wherein the immunogenic composition comprises an immunogenic complex, wherein the immunogenic complex comprises:

32. The method of claim 31, wherein the polypeptide comprising the biotin-binding moiety is a fusion protein comprising:

(i) the biotin-binding moiety; and

(ii) at least one polypeptide antigen.

33. The method of claim 30, wherein the immunogenic composition comprises an immunogenic conjugate, wherein the immunogenic conjugate comprises the polysaccharide antigen covalently conjugated to the polypeptide.

34. The method of any one of claims 30-33, wherein the predetermined target level is at least 2-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

35. The method of any one of claims 30-34, wherein the predetermined target level is at least 5 -fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

36. The method of any one of claims 30-35, wherein the predetermined target level is a level that is at least 10-fold higher than the corresponding level obtained by contacting an APC with an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

37. The method of any one of claims 30-36, wherein the characterizing comprises measuring a level of intracellular polysaccharide antigen present in the APC.

38. The method of any one of claims 30-37, wherein the characterizing comprises measuring a level of polysaccharide antigen associated with the surface of the APC.

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

40. The method of claim 39, wherein the B cell recall response of the immunized subject is characterized in that exposure of the immunized subject to the pathogen produces an antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen at an equivalent or greater level to that produced by the reference composition.

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

42. The method of any one of claims 39-41, wherein the reference composition comprises a polysaccharide antigen that is not associated with a polypeptide antigen.

43. The method of any one of claims 39-41, wherein the reference composition does not comprise a polypeptide antigen expressed by the pathogen.

44. The method of any one of claims 39-41, wherein the reference composition comprises a polysaccharide antigen covalently conjugated to a carrier polypeptide.

45. The method of claim 41, wherein protection against the pathogen comprises a B cell recall response.

46. The method of claim 45, wherein the B cell recall response comprises an antibody response against the polysaccharide antigen induced by exposure of the subject to the pathogen, and wherein the immunogenic composition potentiates the B cell recall response to a level at least 20% higher than the corresponding level produced by administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

47. The method of claim 46, wherein the protection against the pathogen comprises a Thl response.

48. The method of claim 47, wherein the Thl response comprises production of IFN-y and/or TNF-a by CD4+ T cells at a level that is at least 1.1-fold higher than a corresponding level of IFN-y and/or TNF-a produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

49. The method of claim 46, wherein the protection against the pathogen comprises a Th 17 response.

50. The method of claim 49, wherein the Thl7 response comprises production of IL-17, IL- 21, IL-22, IL24 and/or IL-26 by CD4+ T cells at a level that is at least 1.1 -fold higher than a corresponding level of IL- 17, IL-21, IL-22, IL24 and/or IL-26 produced by CD4+ T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

51. The method of claim 46, wherein the protection against the pathogen comprises a CD8 response.

52. The method of claim 51, wherein the CD8 response comprises production of IFN-y, granzyme B, and/or perforin by CD8 T cells at a level that is at least 1.1-fold higher than a corresponding level of IFN-y, granzyme B, and/or perforin produced by CD8 T cells upon administration to the subject of the equivalent dose of an immunogenic composition comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

54. The method of claim 53, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine.

55. The method of claim 53 or 54, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a prime vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein prior to receiving the booster vaccine.

56. The method of any one of claims 53-55, wherein the booster vaccine comprising the polysaccharide antigen is, or comprises, the MAPS vaccine, wherein the MAPS vaccine comprises:

(i) a biotin-binding moiety; and

57. The method of any one of claims 53-55, wherein the booster vaccine comprising the polysaccharide antigen is or comprises a preparation comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

58. The method of any one of claims 53-55, wherein the booster vaccine comprising the polysaccharide antigen is or comprises a polysaccharide antigen covalently conjugated to a carrier polypeptide.

59. The method of any one of claims 53-58, comprising administering the MAPS vaccine to the subject prior to administering the booster vaccine.

(b) a fusion protein comprising:

(i) a biotin-binding moiety; and

61. The method of claim 60, wherein the predetermined target level is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein.

62. The method of claim 60 or 61, wherein the predetermined target level is characterized by production of antibody (e.g., IgG and/or IgM antibody) against the polysaccharide antigen and/or killing of the pathogen by immune sera from the subject in a opsonophagocytic assay (OPA), at a level that is at least 20% higher than the corresponding level of a B cell response in a subject who has received a booster vaccine comprising a polysaccharide antigen of the pathogen that is not associated with a polypeptide antigen, or a polysaccharide antigen of the pathogen covalently conjugated to a carrier protein.

63. The method of any one of claims 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises the MAPS vaccine.

64. The method of any one of claims 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises a preparation comprising a polysaccharide antigen that is not associated with a polypeptide antigen.

65. The method of any one of claims 60-62, wherein the prime vaccine comprising the polysaccharide antigen is or comprises a polysaccharide antigen of covalently conjugated to a carrier polypeptide.

66. The method of any one of claims 60-65, comprising administering the prime vaccine to the subject prior to administering the booster MAPS vaccine.

67. The method of any one of claims 53-66, wherein the B cell response is a T helper (Th)- dependent response against the polysaccharide antigen and/or the polypeptide antigen.

68. The method of any one of claims 1-67, wherein the pathogen is a Streptococcal (e.g., Group A, Group B, and Viridans), Staphylococcal (e.g., S. aureus), Meningococcal, Pneumococcal, Gram-Negative Bacteria (e.g., E. coli, Klebsiella, Pseudomonas, Enterobacter, Citrobacter, Acinetobacter, Serratia, Burkholderia, Salmonella, Shigella, and Bordetella), coronavirus, Mycobacterium (e.g., M. tuberculosis), Plasmodium (e.g., P. falciparum), pathogen.

69. The method of any one of claims 1-68, wherein the immunogenic composition comprises a plurality of different species of immunogenic complexes, wherein the different species comprise different polysaccharide antigens, and/or different polypeptide antigens.

70. The method of any one of claims 1-69, wherein the polysaccharide antigen is or comprises a portion of a capsular polysaccharide of Streptococcus pneumoniae.

71. The method of any one of claims 1-70, wherein the capsular polysaccharide of Streptococcus pneumoniae is selected from: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 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.

72. The method of any one of claims 1-71, wherein the polypeptide antigen is a polypeptide antigen selected from pneumococcal antigens (e.g., Group A, Group B, and Viridans antigens), tuberculosis antigens, anthrax antigens, HIV antigens, seasonal or epidemic flu antigens, Pertussis antigens, Staphylococcus aureus antigens, Meningococcal antigens, Haemophilus antigens, HPV antigens, Shigella antigens, Salmonella antigens, malaria antigens, Pseudomonas antigens, coronavirus antigens, or combinations thereof.

73. The method of any one of claims 1-72, wherein the polypeptide antigen is an SP1500 polypeptide, an SP0785 polypeptide, and/or a pneumolysin polypeptide.

74. The method of any one of claims 1-73, wherein the immunogenic composition is administered as part of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

75. The method of claim 74, wherein the pharmaceutical composition further comprises one or more adjuvants.

76. The method of claim 75, wherein the one or more adjuvants are or comprise a costimulation factor.

77. The method of claim 74 or 75, wherein the one or more adjuvants are selected from the group consisting of aluminum phosphate, aluminum hydroxide, and phosphated aluminum hydroxide.

78. The method of any one of claims 75-77, wherein the one or more adjuvants are or comprise aluminum phosphate.

79. The method of any one of claims 75-78, wherein the pharmaceutical composition is formulated for injection.

80. The method of any one of claims 75-79, wherein the biotin-binding moiety is or comprises a dimeric biotin-binding moiety.

81. The method of any one of claims 75-80, wherein the dimeric biotin-binding moiety is or comprises a rhizavidin polypeptide.

82. The method of any one of claims 75-81, wherein the rhizavidin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or a biotin-binding fragment thereof.

83. The method of any one of claims 1-82, wherein the polypeptide antigen is selected from any one or more of: a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12; a SP0785 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 10 or a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 4.

84. The method of any one of claims 1-83, wherein the polypeptide antigen is a fusion protein, wherein the fusion protein is selected from any one or more of: a fusion protein, comprising, in any order, a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12, fused to a SP0785

I l l polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 10; a fusion protein, comprising, in any order, a SP1500 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 12, fused to a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or a fusion protein, comprising, in any order, a SP0785 polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 10 fused to a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

85. The method of any one of claims 1-84, wherein the polypeptide antigen, or fusion protein is further fused to a rhizavidin polypeptide having an amino acid sequence at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 2.

86. An immunogenic complex comprising:

87. An immunogenic complex comprising:

88. The immunogenic complex of claim 86 or 87, wherein the biotin-binding polypeptide is or comprises avidin.

89. The immunogenic complex of claim 86 or 87, wherein the biotin-binding polypeptide is or comprises rhizavidin, wherein the rhizavidin comprises an amino acid sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 2. The immunogenic complex of any of claims 86-97, wherein the biotin-binding polypeptide further comprises a polypeptide antigen that is a tumor polyscaharride. The immunogenic complex of claims 86-90, wherein the capsular polysaccharide is Streptococcus pneumoniae polysaccharide selected from any of the: serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, HE, HF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48. The immunogenic complex of claims 87-91, wherein the biotin-binding polypeptide further comprises a polypeptide antigen that is a coronavirus antigen. A vaccine composition, comprising two or more species of immunogenic complex according to claims 86 and/or 87, wherein the combined weight of the polysaccharides (PS) and polypeptides in the vaccine contributed by each immunogenic complex is about the same, such that the w/w proteimPS ratio is about 1 : 1 or more than 1: 1. The vaccine composition of claim 93, wherein the w/w protein: PS ratio is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1: 10. The vaccine composition of claim 94, wherein the combined weight of the polysaccharide in the vaccine contributed by the immunogenic complexes ranges from about 0.2pg to about 12 g, or 0.2 g to about 6 g, or 0.2pg to about 20pg, or 0.2pg to about 40pg. A vaccine composition, comprising two or more species of immunogenic complex according to claims 86 and/or 87, wherein the combined weight of the polysaccharides (PS) and polypeptides in the vaccine contributed by each immunogenic complex is different, such that the w/w proteimPS ratio is not about 1: 1. The vaccine composition of claim 96, wherein the w/w protein: PS ratio is 2: 1; 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1. The vaccine composition of claim 96, wherein the combined weight of the polysachharide and polypeptides in the vaccine contributed by each immunogenic complex ranges from about 0.4 pg to about l lOpg. The vaccine composition of any of claims 93-98, comprising at least one immunogenic complex of claim 86 and at least one immunogenic complex of claim 87.

. The vaccine composition of claim 96-99, comprising at least 15 or more species of an immunogenic composition according to claim 87. . The vaccine composition of any of claims 93-100, wherein at least one immunogenic complex comprises a polysaccharide from any of Streptococcus pneumoniae serotypes selected from: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, HA, 11B, 11C, HD, HE, 1 IF, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23 A, 23B, 23F, 24A, 24B, 24F, 25 A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48; and at least one immunogenic complex comprises a polysaccharide selected from any of: meningococcal polysaccharide, a Haemophilus influenze type b polysaccharide, a Streptococcus agalactiae polysaccharide, a Salmonella typhi Vi polysaccharide, a Klebsiella polysaccharide, a Pseudomonas polysaccharide, a Escherichia coli polysaccharide, or a Staphylococcus aureus polysaccharide, Hib (Haemophilus influenzae type B) capsular polysaccharide, meningococcal capsular polysaccharides, the polysaccharide of Bacillus anthracis (the causative agent of anthrax), Neisseria meningitidis (e.g., capsular polysaccharides selected from any of serogroups: A, C, W, W135, or Y). . The vaccine composition of any of claims 93-101, wherein at least one immunogenic complex comprises a polysaccharide from Klebsiella pneumoniae, or a lipopolysaccharide (LPS)-derived polysaccharides, wherein the LPS-derived polysaccharides is a O polysaccharides (OPS) . The vaccine composition of claim 102, wherein the LPS-derived polysaccharides is a core O polysaccharides (COPS), or is selected from, or derived from, an OPS from Klebsiella pneumoniae serotypes selected from: 01, 02, O2ac, 03, 04, 05, 07, 08, or 012, or a CPS from Klebsiella pneumoniae KI, K2, KI 0, KI 6, or KI 9. . The vaccine composition of any of claims 93-103, wherein at least one immunogenic complex comprises a polysaccharide selected from: Type 5 or Type 8 polysaccharide, or any of the polysaccharides or oligosaccharides of Staphylococcus aureus. . The vaccine composition of any of claims 93-104, wherein the vaccine is incorporated into any of: liposome, cochleates, biodegradable polymers, or entrapped in microemulsions. . The vaccine composition of any of claims 93-105, wherein the vaccine is incorporated into any of: liposomes, microemulsion or biodegradable polymer for delivery to the mucosa. . The vaccine composition of any of claims 93-106, wherein the composion further comprises one or more adjuvants, or a mixture of adjuvants. . The vaccine composition of claim 107, wherein the adjuvant is selected from at least one of: aluminium hydroxide, aluminium phosphate (Alum), cholera toxin, lalible toxids, LPS, MPL, or interleukins.

. The vaccine composition of any of claims 93-107, further comprising a nucleic acid encoding a cytokine, e.g., a nucleic acid encoding IL-17 or a cytokine that stimilates IL-17 production. . The vaccine composition of claims 93-109, wherein the vaccine is formulated for administration or delivery to the mucosa. . The vaccine composition of any of claims 93-110, wherein the vaccine composition is lyophilized. . The method of any of claims 1-85, comprising administering a vaccine composition of any of claims 93-111, or an immunogenic complex of any of claims 86 or 87. . A fusion protein, comprising, in any order, a biotin-binding moiety comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO:2, and a PdT polypeptide comprising an amino acid sequence at least 85% or 95% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 4. . A MAPS vaccine for use in the methods of any of claims 1-85, the MAPS vaccine comprising: a biotinylated polysaccharide antigen comprising biotin and a polysaccharide antigen of the pathogen; and (b) a fusion protein of claim 113. The vaccine composition of any of claims 93-111, comprising the MAPS vaccine of claim 114.