Vaccine compositions comprising sialic acid binding domain (SBD) proteins and use thereof to enhance immune response

Abstract

Aspects of the invention described herein relate to a vaccine composition comprising at an antigenic polysaccharide and an immunomodulatory amount of a sialic acid binding moiety, wherein the sialic acid binding moiety comprises sialic acid binding domain (SBD) which is not fused to an antigenic polypeptide.

Description

Attorney Docket: 701039-000134WOPT VACCINE COMPOSITIONS COMPRISING SIALIC ACID BINDING DOMAIN (SBD) PROTEINS AND USE THEREOF TO ENHANCE IMMUNE RESPONSE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 544,119 filed October 13, 2023, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION

[0002] The present invention relates to technologies, compositions, and methods for the prevention and / or treatment of bacterial infections. BACKGROUND OF INVENTION

[0003] Sialic acid is a monosaccharide that is widely present in animals and, to a lesser extent, in microorganisms, including fungi, bacteria and virus. Sialic acid modification is commonly found in glycoproteins and glycolipids in mammalian cells and is important for cell function. However, certain bacteria and virus can incorporate sialic acid onto their surface capsule, such as polysaccharides in the case of bacteria or glycoproteins in the case of virus, so that they can evade host immune defense during infection by using sialic acid (a self-antigen for the host) as a shield. For the same reason, it is difficult to generate immune responses to sialic acid-containing bacterial capsular polysaccharides or viral glycoproteins when used as vaccine targets. In most cases, such sialic acid modification is part of the important epitopes to which antibodies are most effective in functionally removing the target bacterial or viral pathogens.

[0004] Thus, there is a need to develop a method to overcome the “immune suppression” caused by sialic acid modification in bacterial or viral components and induce robust immune responses to these molecules during vaccination. SUMMARY OF THE INVENTION

[0005] The technology disclosed herein relates to a vaccine composition comprising a sialic acid- binding protein domain (SBD) to mask or shield the immune tolerance of a vaccine comprising a pathogenic or antigenic polysaccharide. Moreover, the SBD vaccine compositions disclosed herein comprise any polysaccharide vaccine or composition, that further comprises a SBD as disclosed herein. That is, the inventors have discovered that a sialic acid-binding moiety, e.g., a SBD as disclosed herein, can quench the ability of sialic acids on the surface of bacterial capsular polysaccharides to evade a hose immune system. Accordingly, the technology disclosed herein also relates to a method using a SBD in a vaccine composition to enhance an immune response to a bacterial capsular polysaccharides in the same 1 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT vaccine composition. As disclosed herein, the presence of a sialic acid-binding moiety comprising a SBD protein is that the SBD binds to native sialic acid on the surface of bacterial or other pathogenic polysaccharides and can reduce, or overcome the immune eluding ability of pathogenic polysaccharides to generate an immune response.

[0006] Another aspect of the present invention relates to, methods of making or producing the vaccine composition as disclosed herein, a pharmaceutical composition comprising the immune compositions as disclosed herein and / or a method to induce an immune response in a subject by administering the vaccine composition as disclosed herein.

[0007] One aspect of the technology described herein relates to a vaccine composition comprising at an antigenic polysaccharide and an immunomodulatory amount of a sialic acid binding moiety, wherein the sialic acid binding moiety comprises sialic acid binding domain (SBD) which is not fused to an antigenic polypeptide. In some embodiments, the SBD is selected from any of: SBD1, SBD2, SBD3, SBD4, NanH, NanH2, VcNanH, or a SBD comprising an amino acid sequence that at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of: SEQ ID NO: 1-10.

[0008] In some embodiments, the vaccine composition comprises a SBD is selected from: (i) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 1 (SBD1), or an amino acid having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 (SBD1), or (ii) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 2 (SBD2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 2 (SBD2), or (iii) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 3 (SBD3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 (SBD3), (iv) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 4 (SBD4), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 4 (SBD4), (v) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 5 (NanH), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 5 (NanH), (vi) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 6 (NanH2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 6 (NanH2), (vii) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 7 (NanH3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 7 (NanH3), or (viii) a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 10 (NanH Vibrio Cholera), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 10 (NanH Vibrio Cholera).

[0009] In some embodiments, the SBD is lipidated., e.g., where a lipidated SBD protein comprises at the N-terminus of the SBD protein, a lipidation sequence selected from any of: 2 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT MKKVAAFVALSLLMAGC (SEQ ID NO: 16); MNSKKLCCICVLFSLLAGCAS (SEQ ID NO: 17), MRYSKLTMLIPCALLLSAC (SEQ ID NO: 18), MFVTSKKMTAAVLAITLAMSLSAC (SEQ ID NO: 19) and MIKRVLVVSMVGLSLVGC (SEQ ID NO: 20) or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of SEQ ID NO: 16-20.

[0010] In all aspects disclosed herein, the antigenic polysaccharide present in a vaccine composition as disclosed herein comprises sialic acid on the surface. In alternative embodiments, the antigenic polysaccharide does not comprises sialic acid on the surface. In such embodiments, the antigenic polysaccharide has a negative polarity, e.g., a polarity similar to a pneumococcal polysaccharide.

[0011] In all aspects disclosed herein, the vaccine composition comprises an immunomodulatory amount of a Sialic acid binding moiety that is a ratio, e.g., a ratio of an amount of SBD to the amount of antigenic polysaccharide to suppress the immune tolerance of the antigenic polysaccharide in a subject, as compared to the immune response to the antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety.

[0012] In all aspects disclosed herein, the vaccine composition comprises an immunomodulatory amount of a Sialic acid binding moiety, which comprises a SBD, and is a ratio of an amount of SBD to the amount of antigenic polysaccharide to enhance the immune response of the antigenic polysaccharide in a subject, as compared to the immune response to the antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety. In some embodiments, an immunomodulatory amount of a Sialic acid binding moiety is the amount of SBD to bind to at least 20%, or 30%, or 40%, or 50%, or 60%, or 70% or more than 70% of sialic acids molecules present on the antigenic polysaccharide. In some embodiments, the w / w ratio of SBD protein:antigenic polysaccharide present in the vaccine composition is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 or more than 20:1. In some embodiments, the ratio of SBD to sialic acid molecules present on the antigenic polysaccharide is about 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3.0, 1:3.5, 1:4.0, 1:4.5, 1:5.0 or more than 5:1.

[0013] In all aspects disclosed herein, the vaccine composition comprises an antigenic polysaccharide selected from the group consisting of: polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-positive bacteria; polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-negative bacteria; other bacterial capsular or cell wall polysaccharides; fungal polysaccharides; viral polysaccharides; and polysaccharides derived from cancer or tumor cells. In some embodiments, the antigenic polysaccharide is a capsular polysaccharide from a pathogen and has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

[0014] In some embodiments, the antigenic polysaccharide is a capsular polysaccharide, and in some embodiments, has about 1.0 mM sialic acid per mM of polysaccharide, such as at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM of polysaccharide. In some embodiments, a capsular polysaccharide is selected from any of: Salmonella typhi Vi capsular polysaccharides; 3 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT Salmonella polysaccharides; Shigella polysaccharide, pneumococcal polysaccharides; Haemophili polysaccharides; Meningococcal polysaccharides; Staphylococcus aureus polysaccharides; Bacillus anthracis polysaccharides; Streptococcus polysaccharides; Pseudomonas polysaccharides; Cryptococcus polysaccharides; and viral glycoproteins.

[0015] In some embodiments, the vaccine composition can comprise a polysaccharide that exists in a polysaccharide-protein conjugate, including, but not limited to, immunogenic polysaccharide-protein conjugates where the protein is covalently bound to the polysaccharide or non-covalently associated with the polysaccharide. In some embodiments, the vaccine composition comprises a polysaccharide vaccines which is a multiple antigen presenting system (MAPS), as disclosed in any of patent applications: WO / 2020 / 056127, WO / 2020 / 056202, WO / 2014 / 124228, WO / 2023 / 039223, US11560410B2, WO / 2018 / 183475, WO / 2018 / 217564, WO / 2023 / 102359, WO / 2023 / 102359A9, WO / 2012 / 155007, WO / 2023 / 192997A2WO / 2013 / 134656, WO / 2023 / 039108, WO / 2012 / 155053, WO / 2023 / 172741A2, WO / 2014 / 124228, WO / 2020 / 056127, WO / 2017 / 192801, WO / 2020 / 056202, WO / 2018 / 237221, WO / 2023 / 039223, US11305001, US20220362367, US20210008192, US20200121777, US20140154287, US10766932, US20210332090, US20230233667, US11560410, US10611805, US20160090404, US10017548, US9499593, US20140154286, US20200407404, US20190119335, US20150374811, US11576958, US20230081705, US20230089151, US20200087361, US20190119332, US11013793, US11701416, US20210346487, US20200222522, US11612647, US20220072118, US20230091255, or US20150374811, or WO2023 / 102359, each of which is incorporated herein in its entirety by reference, or International applications: PCT / 2023 / 76822 and PCT / 2023 / 76878, and PCT / US2023 / 76909, filed on October 14, 2023, each of which is incorporated herein in its entirety by reference.

[0016] Another aspect of the technology disclosed herein relates to uses of the vaccine composition to induce an immune response to a polysaccharide in a subject. Another aspect of the technology disclosed herein relates to a method to induce an immune response in a subject comprising administering the vaccine composition of any of claims 1-19, wherein the immune response to the polysaccharide in the vaccine is greater as compared to the immune response to the same polysaccharide in the absence of the presence of a sialic acid binding protein (SBD). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG.1 is a schematic drawing that shows an exemplary antigenic polysaccharide that comprises a plurality of sialic acids, and a vaccine comprising the combination of an antigenic polysaccharide and a SBD protein, where the SBD protein binds to, or associates with the sialic acids on the polysaccharide, thereby masking or quenching their ability to evade the immune response.

[0018] FIG.2 shows that SBD as a stand-along protein enhances an immune response to a MAPS- GBS immunogenic complex comprising a polysaccharide from GBS subtypes 1b, II, and III, as compared to Rhavi, which serves as a carrier protein. 4 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0019] The technology disclosed herein relates to a vaccine composition comprising a sialic acid- binding protein domain (SBD) to mask or shield the immune tolerance of a vaccine comprising a pathogenic or antigenic polysaccharide. More specifically, the technology disclosed herein relates to the use of a sialic acid-binding domain (SBD) to overcome the immune suppression of native sialic acid on the surface of a polysaccharide, e.g., a polysaccharide with sialic acids on the surface, such as but not limited to GBS polysaccharides. Surprisingly, the inventors have also discovered that use of a sialic acid-binding domain (SBD) can also increase the immunogenicity of a plurality of different polysaccharide, even if the polysaccharide does not comprise sialic acid molecules on it surface.

[0020] In some embodiments, and as an added benefit, the non-covalent association of the SBD to a sialic acid on the antigenic polysaccharide blocks the immune suppressive function of sialic acids on an antigenic polysaccharide. That is, without being limited to theory, a sialic acid-binding domain (SBD) can bind to a sialic acid present on the surface of a polysaccharide and essentially mask (e.g., shield) the immune tolerance of the antigenic polysaccharide. Stated differently, the binding of the SBD protein to sialic acid on a polysaccharide reduces the hosts’ exposure to the sialic acids, which are normally recognized as a self-antigen by the host. With reduced exposure due to the masked sialic acid molecules, the host recognizes the polysaccharide as foreign and thus the binding of SBD fusion protein on the polysaccharide increases the immunogenicity of the polysaccharide to the host. Herein the inventors have demonstrated that a SBD polypeptide as disclosed herein can quench the ability of sialic acids on the surface of capsular polysaccharides to evade a host immune system, and therefore improve the host’s immune response to the polysaccharide when administered to the subject. II. SBD proteins

[0021] A sialic acid binding domain (SBD) useful as a component of a vaccine composition as disclosed herein exhibits an affinity for sialic acid—including all forms of sialic acid described above and, in particular sialic acid present on the surface of mammalian cells. For example, the molecules of this invention may exhibit an affinity for cell membrane receptors, which comprise sialic acid. Cell receptors of this type may be present on the surface of epithelial cells—including epithelial cells of the mucosal and respiratory tracts. A number of pathogens, including viral and / or bacterial pathogens may express molecules, which exhibit an affinity for sialic acid. Pathogens of this type have evolved to exploit cell surface sialic acid moieties as a means to bind to host cells. Once bound to a cell via, for example a host cell surface bound sialic acid moiety, a pathogen may colonise the cell surface and / or infect / enter the cell.

[0022] In particular embodiments, a sialic acid binding domain (SBD) that is a component of a vaccine composition as disclosed herein exhibits affinity for sialic acid present on an antigenic polysaccharide. In particular embodiments, a sialic acid binding domain (SBD) that is a vaccine 5 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT composition as disclosed herein can also exhibits affinity for an antigenic polysaccharide that does not comprise a sialic acid, wherein the antigenic polysaccharide has a negative polarity, and / or has a similar polarity to s. pneumococci CP1 polysaccharide.

[0023] In some embodiments, a SBD of a vaccine composition as disclosed herein may exhibit an affinity for α-2,6-linked sialic acid receptors predominantly present on cells of the human upper respiratory tract. Additionally or alternatively, the sialic acid binding receptors may exhibit an affinity for α-2,3-linked sialic acid receptors present on cells of the upper and lower respiratory tracts.

[0024] As used herein, a “sialic acid binding domain (SBD)” or “sialic acid binding molecule (SBM)” are used interchangeably, and refers to a portion, fragment of variant of a sialic acid binding protein that binds or has affinity for a sialic acid moiety on the surface of a polysaccharide. It should be understood that any polypeptide or molecules which exhibit an affinity for sialic acid, bind to or otherwise couple to or associate with sialic acid moieties is encompassed in the term SBD. Thus the term “sialic acid binding molecule” may encompass any fragment, which retains an ability to bind to or otherwise couple or associate with a sialic acid moiety.

[0025] In some embodiments, a SBD as disclosed herein may comprise a single molecule capable of binding sialic acid (a monomeric or monovalent molecule, for example) or, alternatively, two or more sialic acid binding molecules (which may all be the same or different—a polymeric or multivalent molecule, for example). a) Exemplary Sialic acid binding domain (SBD):

[0026] In some embodiments, a SBD comprises one or more of the polypeptides listed in Table 1.

[0027] Table 1. Exemplary SBD proteins for use in the vaccine compositions.6 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0028] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises, a SBD selected from any of SBD1, SBD2, SBD3, SBD4, NanH, NanH2, VcNanH, or a protein comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 1-10 or a protein having an amino acid sequence with at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of: SEQ ID NOs: 1-10.

[0029] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 1, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 (SBD1).

[0030] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 2, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 2 (SBD2).

[0031] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 3, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 (SBD3).

[0032] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 4, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 4 (SBD4).

[0033] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 5, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 5 (NanH Salmonella).

[0034] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 6, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 6 (NanH2 Salmonella).

[0035] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 7, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 7 (NanH3 Trypanosoma).

[0036] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 8, or a functional variant thereof 7 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 8 (SBD1-full length (NanA-full length).

[0037] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 9 (NanH-full length (Vibrio cholera).

[0038] In some embodiments, a SBD protein useful in a vaccine composition as disclosed herein comprises a protein with an amino acid sequence of SEQ ID NO: 10, or a functional variant thereof comprising an amino acid sequence having at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 10 (NanH-truncated (Vibrio cholera).

[0039] A SBD of a vaccine composition as disclosed herein can comprise one or more moieties that exhibit an affinity for sialic acid.

[0040] (i) SBD1 (NanA): In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of Streptococcus pneumoniae NanA sialidase (NanA), where the amino acid sequence is SEQ ID NO: 1 (1035 amino acids) and has been deposited under accession number P62575. In some embodiments, a SBD protein as disclosed herein comprises amino acids 121- 305 the full length NanA polypeptide of amino acids of SEQ ID NO: 8. In some embodiment, a SBD as disclosed herein comprises an amino acid sequence of SEQ ID NO: 1 (180aa), or an functional variant or fragment thereof.

[0041] In some embodiments, a SBD in a vaccine composition as disclosed herein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of a SBD polypeptide of SEQ ID NO: 1. In some embodiments, a SBD protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of the sequence shown in SEQ ID NO: 1. In some embodiments, a SBD polypeptide is a SBD1 polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of the sequence shown in SEQ ID NO: 1.

[0042] (ii) SBD2 (NanL): In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of NanL sialidase (NanL). In some embodiments, a SBD is a fragment of NanL and comprises acids 81-272 the full length NanL polypeptide. In some embodiment, a SBD for use in a vaccine composition as disclosed herein comprises an amino acid sequence of SEQ ID NO: 2 (192aa), or an immunogenic variant or fragment thereof.

[0043] In some embodiments, a SBD protein comprises 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, 180, 185 or 191 consecutive 8 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT amino acids of a SBD2 polypeptide of SEQ ID NO: 2. In some embodiments, a SBD protein comprises 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, 180, 185 or 191 consecutive amino acids of the sequence shown in SEQ ID NO: 2. In some embodiments, a SBD polypeptide is a SBD2 immunogenic polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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, 180, 185 or 191 consecutive amino acids of SBD2 amino acid sequence shown in SEQ ID NO: 2.

[0044] (iii) SBD3 (NanB): In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of NanB sialidase (NanB). In some embodiments, a SBD is a fragment of amino acids 40-230 the full length NanB polypeptide. In some embodiment, a SBD for use in a vaccine composition as disclosed herein comprises an amino acid sequence of SEQ ID NO: 5 (191aa), or an functional variant or fragment thereof.

[0045] In some embodiments, a SBD protein comprises 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, 180, 185, 190 or 192 consecutive amino acids of a SBD3 polypeptide of SEQ ID NO: 3. In some embodiments, a SBD protein comprises 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, 180, 185, 190 or 192 consecutive amino acids of the sequence shown in SEQ ID NO: 3. In some embodiments, a SBD polypeptide useful in a vaccine composition as disclosed herein is a SBD3 polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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, 180, 185, 190 or 192 consecutive amino acids of SBD3 amino acid sequence shown in SEQ ID NO: 3.

[0046] (iv) SBD4 (NanC): In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of NanC sialidase (NanC). In some embodiments, a SBD is a fragment of amino acids 82-270 the full length NanC polypeptide. In some embodiment, a SBD for use in a vaccine composition as disclosed herein as disclosed herein comprises an amino acid sequence of SEQ ID NO: 4 (189aa), or an functional variant or fragment thereof.

[0047] In some embodiments, a SBD protein comprises 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, 180, 185, 190 or 192 consecutive amino acids of a SBD4 polypeptide of SEQ ID NO: 6. In some embodiments, a SBD protein comprises 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, 180, 185, 187 or 189 consecutive amino acids of the sequence shown in SEQ ID NO: 4. In some embodiments, a SBD polypeptide is a SBD4 immunogenic polypeptide and comprises an amino 9 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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, 180, 185, 187 or 189 consecutive amino acids of SBD4 amino acid sequence shown in SEQ ID NO: 4.

[0048] (v) NanH: In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of Salmonella NanH sialidase (NanH). In some embodiments, a SBD is a fragment of the full length NanH polypeptide. In some embodiment, a SBD protein as disclosed herein comprises NanH having an amino acid sequence of SEQ ID NO: 5 (381aa), or an functional variant or fragment thereof.

[0049] In some embodiments, a SBD protein comprises 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 380 consecutive amino acids of a NanH polypeptide of SEQ ID NO: 5. In some embodiments, a SBD protein comprises 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 380 consecutive amino acids of a NanH polypeptide of SEQ ID NO: 5. In some embodiments, a SBD protein is a NanH immunogenic polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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 380 consecutive amino acids of a NanH polypeptide of SEQ ID NO: 5.

[0050] (vi) NanH2: In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of Salmonella NanH2 sialidase (NanH2). In some embodiments, a SBD is a fragment of the full length NanH2 polypeptide. In some embodiment, a SBD for use in a vaccine composition as disclosed herein as disclosed herein comprises NanH2 having an amino acid sequence of SEQ ID NO: 8 (404aa), or an functional variant or fragment thereof.

[0051] In some embodiments, a SBD protein comprises 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 380 consecutive amino acids of a NanH2 polypeptide of SEQ ID NO: 6. In some embodiments, a SBD protein comprises 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 380 consecutive amino acids of a NanH2 polypeptide of SEQ ID NO: 6. In some embodiments, a SBD protein is a NanH2 immunogenic polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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 380 consecutive amino acids of a NanH2 polypeptide of SEQ ID NO: 6. 10 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0052] (vii) NanH3: In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of NanH3 sialidase (NanH3) from Trypanosoma cruzi. In some embodiments, a SBD is a fragment of amino acids 4-399 the full length NanH3 polypeptide. In some embodiment, a SBD for use in a vaccine composition as disclosed herein comprises an amino acid sequence of SEQ ID NO: 7 (396aa), or an functional variant or fragment thereof.

[0053] In some embodiments, a SBD protein comprises 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, 380, 390 or 396 consecutive amino acids of a NanH3 polypeptide of SEQ ID NO: 7. In some embodiments, a SBD protein comprises 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, 350380, 390 or 396 consecutive amino acids of a NanH3 polypeptide of SEQ ID NO: 7. In some embodiments, a SBD protein is a NanH3 polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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, 350380, 390 or 396 consecutive amino acids of a NanH3 polypeptide of SEQ ID NO: 7.

[0054] (viii) NanH (Vibro cholera) (VcNaNH): In some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of NanH sialidase from Vibro cholera. In some embodiments, a SBD is a fragment of amino acids 25-216 the full length NanH (Vibro cholera) polypeptide which comprises the amino acids of SEQ ID NO: 9 (781aa). In some embodiment, a SBD for use in a vaccine composition as disclosed herein comprises an amino acid sequence of SEQ ID NO: 10 (192aa), or an functional variant or fragment thereof.

[0055] In some embodiments, a SBD protein comprises 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, 160, 170, 180, or 190 or 192 consecutive amino acids of a NanH (Vibro cholera) polypeptide of SEQ ID NO: 10. In some embodiments, a SBD protein comprises 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, 160, 170, 180, or 190 or 192 consecutive amino acids of a NanH (Vibro cholera) polypeptide of SEQ ID NO: 10. In some embodiments, a SBD protein is a NanH (Vibro cholera) polypeptide and comprises an amino acid sequence that has at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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, 160, 170, 180, or 190 or 192 consecutive amino acids of a NanH (Vibro cholera) polypeptide of SEQ ID NO: 10.

[0056] Similar or homologous sialic acid binding modules present in other organisms are to be encompassed within the scope of the term “SBD” herein. In some embodiments, additional exemplary SBD for use in the fusion proteins as disclosed herein comprise the sialic acid binding domain (SBD) of 11 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT Vibrio cholerae NanH sialidase (VcNanH). Accordingly, in some embodiments, an exemplary SBD for use in a vaccine composition as disclosed herein is a fragment of Vibrio cholerae NanH sialidase (VcNanH sialidase), where the amino acid sequence is deposited under accession number A5F7A4 and is as SEQ ID NO: 9 (781 amino acids). In some embodiments, a SBD region of VcNanA is from amino acid residue 25 to 216 of SEQ ID NO: 9, and corresponds to amino acid sequence SEQ ID NO: 10.

[0057] In some embodiments, a SBD for use in a SBD protein is a protein or binding moiety which binds to a modified sialic acid, where modifications of sialic acids include diverse forms differing in position 5 of an amino group of neuraminic acid derivatives or an hydroxyl group of 3-deoxy-D- glycero-D-galactononulosonic acid (Kdn), different acylations of the NH2at position 5 (glycolyl, acetyl), and various substituent of the different hydroxyl groups including phosphate, sulfate, methyl, acetyl, etc. >50 different derivatives of sialic acids are disclosed in Table 1 of Ghosh, S. (2020). Sialic acids and sialoglycoconjugates in the biology of life, health and disease. Academic Press, which is incorporated herein in its entirety. Two most commonly expressed members of sialic acid family are Neu5Ac and Neu5Gc followed by KDN (2-keto-3-deoxy-nononic acid) and Neu (neuraminic acid). Accordingly, a SBD for use in a SBD protein is a protein or binding moiety which has affinity for, and binds to any of: Neu5Ac, Neu5Gc, KDN, Neu. In some embodiments, a SBD for use in a SBD protein is a protein or binding moiety which has affinity for sialic acid that have modifications to core structures of sialic acid, including modifications such as O-acetylation, O-methylation, or introduction of O-lactyl groups, sulfate, or phosphate esters at positions 4, 7, 8, and / or 9.

[0058] In some embodiments, a SBD protein as described herein, or disclosed in Table 1 can be modified or covalently bound to another molecule, where the molecule is not an antigenic protein. This additional molecule may, for example, increase the half-life, solubility, bioavailability, or immunogenicity of the fusion protein. Molecules that may be covalently bound to the SBD protein include a carbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, ranging from molecular weights of below 300 g / mol to over 10,000,000 g / mol. PEG chains can be linear, branched, or with comb or star geometries. In some embodiments, the fusion protein is covalently bound to a moiety that stimulates the immune system. An example of such a moiety is a lipid moiety. In some instances, lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innate immune system. IV. Antigenic polysaccharides for use in the vaccine composition

[0059] One component of a vaccine composition as disclosed is an antigenic or immunogenic polysaccharide (PS).In some embodiments, the vaccine can optionally, comprise additional elements that do not negatively impact the antigenic polysaccharide’s function of (i) inducing an immune response to the polysaccharide and (ii) presenting at least one polypeptide antigen(s) to the immune 12 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT system in immunogenic fashion. In some embodiments, the immunogenic polysaccharide is a synthetic polysaccharide.

[0060] It is envisioned that the polysaccharide used in a vaccine composition as disclosed herein is immunogenic, that is, it helps induce a specific immune response, and herein is referred to as an “immunogenic polysaccharide” or “antigenic polysaccharide”. The specific immune response recognizes the particular immunogenic PS and provides a response to the immunogenic complex, and as explained herein, the response includes both a humoral and cell-mediated response.

[0061] As used herein, the term "saccharide" refers to a single sugar moiety or monosaccharide unit as well as combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The term "saccharide" may be used interchangeably with the term "carbohydrate." The polysaccharide may be linear or branched.

[0062] A "monosaccharide" as used herein refers to a single sugar residue in an oligosaccharide. The term "disaccharide" as used herein refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond. In one embodiment, the polysaccharide is an oligosaccharide (OS). An "oligosaccharide" as used herein refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, an individual monomer unit or moiety is a monosaccharide which is, or can be, bound through a hydroxyl group to another monosaccharide unit or moiety. Oligosaccharides can be prepared by either chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides may be prepared by in vitro enzymatic methods.

[0063] In a preferred embodiment, the polysaccharide of a vaccine composition as disclosed herein is a polysaccharide (PS), which refers to a linear or branched polymer of at least 5 monosaccharide units or moieties. For clarity, larger number of repeating units, wherein n is greater than about 5, such as greater than about 10, will be referred to herein as a polysaccharide.

[0064] In one embodiment, the polysaccharide is a cell surface polysaccharide. A cell surface polysaccharide refers to a polysaccharide having at least a portion located on the outermost bacterial cell membrane or bacterial cell surface, including the peptidoglycan layer, cell wall, and capsule. Typically, a cell surface polysaccharide is associated with inducing an immune response in vivo. A cell surface polysaccharide may be a "cell wall polysaccharide" or a "capsular polysaccharide." A cell wall polysaccharide typically forms a discontinuous layer on the bacterial surface.

[0065] In one embodiment, the polysaccharide is a capsular polysaccharide. A capsular polysaccharide refers to a glycopolymer that includes repeating units of one or more monosaccharides joined by glycosidic linkages. A capsular polysaccharide typically forms a capsule-like layer around a bacterial cell. "Capsular polysaccharide" or "capsule polysaccharide" refers to the polysaccharide capsule that is external to the cell wall of most bacterial isolates. 13 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0066] In some embodiments, the polysaccharide of a vaccine composition as disclosed herein is a naturally occurring polysaccharide, e.g., a polysaccharide derived or purified from a pathogen, e.g., including, but not limited to, bacterial cells, and can be, for example, a capsular or noncaspular PS. In some embodiments, the polysaccharide is derived or purified from eukaryotic cells, e.g., fungi, insect or plant cells. In yet other embodiments, the polysaccharide is derived from mammalian cells, such as virus-infected cells or cancer cells. In general, such immunogenic polysaccharides are well known in the art and are encompassed for use in a vaccine composition as disclosed herein. (a) sialic acid polysaccharides:

[0067] In some embodiments, a vaccine composition as disclosed herein comprises a polysaccharide that comprises a sialic acid. The term “sialic acid” as used herein, embraces all forms of N- or O- substituted neuraminic acid and includes all synthetic, naturally occurring and / or modified forms thereof. Sialic acids may be found as components of cell surface molecules, glycoproteins and glycolipids. Most often, sialic acids are present at the end (terminal regions) of sugar chains connected to cell membranes and / or proteins. The sialic acid family encompasses a number (approximately 50) of derivatives that may result from acetylation, glycolylation, lactonisation and methylation at C4, C5, C7, C8 and C9. All such derivatives are to be embraced by the term “sialic acid”

[0068] Furthermore, sialic acids are found linked α(2,3) or α(2,6) to Gal and GalNAc or α(2,8) or α(2,9) to another sialic acid. Accordingly, it is important to understand that while the term “sialic acid” is used throughout this specification, it encompasses all derivatives, analogues or variants (either naturally occurring or synthetically generated) thereof as well as monomers, dimers, trimers, oligomers, polymers or concatamers comprising the same.

[0069] In some embodiments, the immunogenic polysaccharide is sialylated polysaccharide, e.g., comprises at least one sialic acid moiety, as disclosed herein. In some embodiments, the native level of sialic acid on a polysaccharide is modified, e.g., the percentage increased and / or decreased. In one embodiment, a capsular polysaccharides present in a vaccine composition as disclosed herein can comprise their natural sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharides may be desialylated up to about 40% (sialylation level greater than about 60%), such as up to about 35% (sialylation level greater than about 65%), up to about 30% (sialylation level greater than about 70%), up to about 25% (sialylation level greater than about 75%), up to about 20% (sialylation level greater than about 80%), up to about 15% (sialylation level greater than about 85%), up to about 10% (sialylation level greater than about 90%), and up to about 5% (sialylation level greater than about 95%).

[0070] It should be noted that 100% sialic acid level corresponds to about 1.0 mM sialic acid per mM of polysaccharide. Therefore, the capsular polysaccharides may have about 1.0 mM sialic acid per mM of polysaccharide, such as at least about 0.95 mM sialic acid per mM of polysaccharide. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM of polysaccharide, such as at least about 0.65 mM sialic acid per mM of polysaccharide, at least about 0.7 14 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT mM sialic acid per mM of polysaccharide, at least about 0.75 mM sialic acid per mM of polysaccharide, at least about 0.8 mM sialic acid per mM of polysaccharide, at least about 0.85 mM sialic acid per mM of polysaccharide, at least about 0.9 mM sialic acid per mM of polysaccharide, or at least about 0.95 mM sialic acid per mM of polysaccharide.

[0071] The terminal sialic residues of some capsular polysaccharide (CP) serotypes are partially O- acetylated (OAc) (Lewis, A.L., et al., Proceedings of the National Academy of Sciences USA, 101 (30): 11123-8 (2004)). GBS Serotypes lb, III, IV, V, VI, and IX are partially O- acetylated (up to -40%), whereas serotypes la, II, and VII have little or no O-acetylation (less than about 5%) (Lewis 2004). In one embodiment of the invention, the capsular polysaccharides comprise their natural O-acetylation level (about 0% to about 40%). In another embodiment, the capsular polysaccharides may be de-O- acetylated (less than about 5%). The degree of O-acetylation of the polysaccharide or oligosaccharide can be determined by any method known in the art, for example, by proton NMR (Lemercinier, X., et al., Carbohydrate Research, 296:83-96 (1996); Jones, C, et al., Journal of Pharmaceutical and Biomedical Analysis, 30: 1233-1247 (2002); Int'l Patent Appl. Pub. Nos. WO 2005 / 033148 and WO 00 / 56357). Another commonly used method is described by Hestrin, S., J. Biol. Chem., 180:249-261 (1949).

[0072] It should also be noted that 100% O-acetate corresponds to about 1.0 mM O- acetate per mM of saccharide repeating unit. Accordingly, partially O-acetylated polysaccharides compr2se at least about 0.1 , 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM saccharide repeating unit. A de-O- acetylated polysaccharide comprises less than about 0.01 , 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM saccharide repeating unit.

[0073] In some embodiments, a vaccine composition as disclosed herein comprises a polysaccharide that comprises one or more natural sialic acid family members selected from any of those listed in Table 2:

[0074] Table 2:15 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT16 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT(b) Poly N-Acetylated Glucosamine (PNAG) polysaccharides: In some embodiments, a polysaccharide in a vaccine composition as disclosed herein comprises PNAG. PNAG is a polysaccharide intercellular adhesion and is composed of a polymer of β-(1→6)-linked glucosamine, optionally substituted with N- acetyl and / or O-succinyl constituents. This polysaccharide is present in both S. aureus and S. epidermidis and can be isolated from either source (Joyce et al 2003, Carbohydrate Research 338; 903; Maira-Litran et al 2002, Infect. Imun.70; 4433). For example, PNAG may be isolated from S. aureus strain MN8m (WO 04 / 43407). The preparation of dPNAG is described in WO 04 / 43405, which is incorporated herein in its entirety by reference. The term PNAG as used herein also encompasses a polysaccharide previously known as poly-N-succinyl-β-(1→6)-glucosamine (PNSG) (Maira-Litran et al 2002, Infect. Imun.70; 4433). 17 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0075] PNAG may be of different sizes varying from over 400 kDa to between 75 and 400 kDa to between 10 and 75 kDa to oligosaccharides composed of up to 30 repeat units (of (β-(1→6)-linked glucosamine, optionally substituted with N-acetyl and O-succinyl constituents). Any size of PNAG polysaccharide or oligosaccharide may be used in a vaccine composition of the invention, for example a size of over 40 kDa can be used. Sizing may be achieved by any method known in the art, for instance by microfluidisation, ultrasonic irradiation or by chemical cleavage (WO 03 / 53462, EP497524, EP497525). Size ranges of PNAG are for example 40-400 kDa, 50-350 kDa, 40-300 kDa, 60-300 kDa, 50-250 kDa and 60-200 kDa. PNAG can have different degree of acetylation due to substitution on the amino groups by acetate. PNAG produced in vitro is almost fully substituted on amino groups (95- 100%). Alternatively, a deacetylated PNAG can be used having less than 50%, 40%, 30%, 20%, 10% or 5% N-acetylation. Use of a deacetylated PNAG allows opsonic killing of Gram positive bacteria, optionally S. aureus and / or S. epidermidis (WO 04 / 43405). In an embodiment, the PNAG has a size between 40 kDa and 300 kDa and is deacetylated so that less than 50%, 40%, 30%, 20%, 10% or 5% of amino groups are N acetylated. In an embodiment, the PNAG is not O-succinylated or is O-succinylated on less than 25, 20, 15, 10, 5, 2, 1 or 0.1% of residues. The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide or oligosaccharide in which less than 50%, 40%, 30%, 20%, 10% or 5% of the amino groups are acetylated.

[0076] As used herein, the term PNAG encompasses both acetylated and deacetylated forms of the saccharide. In an embodiment, PNAG is deacetylated to form dPNAG, by chemically treating the native polysaccharide. For example, the native PNAG is treated with a basic solution such that the pH rises to above 10. For instance, the PNAG is treated with 0.1-5M, 0.2-4M, 0.3-3M, 0.5-2M, 0.75-1.5M or 1M NaOH, KOH or NH4OH. Treatment is for at least 10 or 30 minutes, or 1, 2, 3, 4, 5, 10, 15 or 20 hours at a temperature of 20-100, 25-80, 30-60 or 30-50 or 3545° C. dPNAG may be prepared as described in WO 04 / 43405.

[0077] In some embodiments, a polysaccharide in a vaccine composition as disclosed herein is a biotinylated polysaccharide. In some embodiments, a polysaccharide in a vaccine composition as disclosed herein does not natively comprise a sialic acid has a general positive polarity.

[0078] In some embodiments, a vaccine composition as disclosed herein described herein includes one or more polysaccharide is selected from a polysaccharide from the group consisting of: S. aureus, Vi polysaccharide, pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharide, Haemophilus influenzae Type b polysaccharide, Meningococcal polysaccharide, 0- antigens from Gram-negative bacteria and other bacterial capsular or cell wall polysaccharides. In some embodiments, a vaccine composition as disclosed herein comprises an immunogenic polysaccharide selected from type 1 capsular polysaccharide (CP1) of Streptococcus pneumoniae, type 5 capsular polysaccharide (CP5) of S. aureus or type 8 capsular polysaccharide (CP8) of S. aureus.

[0079] Staphylococcal microorganisms capable of causing invasive disease generally also are capable of producing a capsule polysaccharide (CP) that encapsulates the bacterium and enhances its resistance 18 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT to clearance by the host innate immune system. The CP serves to cloak the bacterial cell in a protective capsule that renders the bacteria resistant to phagocytosis and intracellular killing. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are frequently an important virulence factor for many bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae and Group B streptococci.

[0080] In some embodiments, a polysaccharide for use in a vaccine composition as disclosed herein can comprises N. meningitidis capsular polysaccharides from at least one, two, three or four of the serogroups A, C, W, W135, or Y. In some embodiments, a polysaccharide for use in a vaccine composition as disclosed herein is selected from the group consisting of: Salmonella typhi Vi capsular polysaccharide, pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharide, Haemophilus influenzae Type b (Hibb) capsular polysaccharide, Haemophili polysaccharide, Meningococcal polysaccharide, polysaccharides or oligosaccharides from Gram-positive bacteria (e.g., Staphylococcus aureus capsular polysaccharide, Bacillus anthracis polysaccharide), Streptococcus polysaccharides (e.g., Gp A and Gp B), Pseudomonas polysaccharide, fungal polysaccharides (e.g., cryptococcys polysaccharides), viral polysaccharides (e.g., glycoprotein) and other bacterial capsular or cell wall polysaccharides. In some embodiments, an immunogenic polysaccharide is selected from any of the following, dextran, Vi polysaccharide of Salmonella typhi, pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide (CWPS), meningococcal polysaccharide, Haemophilus influenzae type b polysaccharide, or any another polysaccharide of viral, prokaryotic, or eukaryotic origin.

[0081] It is important that a Capsule Polysaccharide (CP) a polysaccharide for use in a vaccine composition as disclosed herein is immunogenic. The molecular weight of a capsule polysaccharides is an important consideration, as a high molecular weight capsule polysaccharide can induce certain antibody immune responses due to a higher valency of the epitopes present on the antigenic surface. In some embodiments, a CP8 or CP5 used in a vaccine composition as disclosed herein is a high molecular weight capsule polysaccharide type 5 (CP5) and type 8 (CP8) from S. aureus or a CP1 of Streptococcus pneumonia, CP1) of Streptococcus pneumoniae, however, it is envisioned that any high molecular weight capsule polysaccharide from a pathogen can be used as a polysaccharide in a vaccine composition as disclosed herein .

[0082] In some embodiments, the immunogenic polysaccharide for use in a vaccine composition as disclosed herein is a branched polymer. In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein is a single chain polymer.

[0083] In some embodiments, a polysaccharide for use in a vaccine composition as disclosed herein are exemplified in Table 3:

[0084] Table 3. Example polysaccharides present in a vaccine composition as disclosed herein19 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0085] In some embodiments, the immunogenic polysaccharide for use in the vaccine composition as disclosed herein comprises at least 10 carbohydrate repeating units, or at least 20, or at least 50, or at 20 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT least 75, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or more than 500 repeating units, inclusive.

[0086] In one aspect of the invention, the immunogenic polysaccharide (PS) for use in the vaccine composition as disclosed herein can have a molecular mass of <500 kDa or >500 kDa. In another aspect of the invention, the PS has a molecular mass of <70 kDa. In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein is a large molecular weight polymer, e.g., a polymer can be of an average molecular weight of between about 425-500 kDa, inclusive, for example, at least 300 kDa, or at least 350 kDa, or at least 400 kDa, or at least 425 kDa, or at least 450 kDa, or at least 500 kDa or greater than 500 kDa, inclusive, but typically less than 500 kDa. In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein can be a small molecular weight polymer, e.g., a polymer can be of an average molecular weight of between about 60 kDA to about 90 kDa, for example, at least 50 kDa, or at least 60 kDa, or at least 70 kDa, or at least 80 kDa, or at least 90 kDa, or at least 100 kDa, or greater than 100 kDa, inclusive, but generally less than about 120 kDa.

[0087] In some embodiments, the immunogenic polysaccharide for use in the vaccine composition as disclosed herein is harvested and purified from a natural source; and in other embodiments, the polysaccharide is synthetic. Methods to produce synthetic polymers, including synthetic polysaccharides, are known to persons of ordinary skill and are encompassed in the compositions and methods as disclosed herein.

[0088] In some embodiments, a type 5 and / or type 8 capsular polysaccharide or oligosaccharide included in a vaccine compositions as disclosed herein has a molecular weight of between 20 kDa and 1000 kDa. In some embodiments, the type 5 and / or type 8 and / or type 1 capsular polysaccharide or oligosaccharide of a vaccine compositions as disclosed herein has a molecular weight of between 200 kDa and 5000 kDa, or a molecular weight range of between 70 kDa and 300 kDa, or a molecular weight range of between 500 kDa and 2500 kDa.

[0089] High molecular weight capsular polysaccharides are able to induce certain antibody immune responses due to a higher valence of the epitopes present on the antigenic surface. The isolation of “high molecular weight capsular polysaccharides” is contemplated for use in the compositions and methods of the present invention. In some embodiments, high molecular weight serotype 5 or 8 capsular polysaccharide can be isolated and purified ranging from 20 kDa to 1000 kDa in molecular weight. In one embodiment, high molecular weight serotype 5 or 8 capsular polysaccharide can be isolated and purified ranging from 50 kDa to 700 kDa in molecular weight, or ranging from 50 kDa to 300 kDa in molecular weight, or ranging from 70 kDa to 300 kDa, or ranging from 90 kDa to 250 kDa, or ranging from 90 kDa to 150 kDa in molecular weight, or ranging from 90 kDa to 120 kDa in molecular weight, or ranging from 80 kDa to 120 kDa in molecular weight. In some embodiments, a type 1, and / or type 5 and / or type 8 capsular polysaccharide or oligosaccharide included in a vaccine compositions as disclosed herein has a high molecular weight of any of 70 kDa to 100 kDa in molecular weight; 70 kDa 21 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT to 110 kDa in molecular weight; 70 kDa to 120 kDa in molecular weight; 70 kDa to 130 kDa in molecular weight; 70 kDa to 140 kDa in molecular weight; 70 kDa to 150 kDa in molecular weight; 70 kDa to 160 kDa in molecular weight; 80 kDa to 110 kDa in molecular weight; 80 kDa to 120 kDa in molecular weight; 80 kDa to 130 kDa in molecular weight; 80 kDa to 140 kDa in molecular weight; 80 kDa to 150 kDa in molecular weight; 80 kDa to 160 kDa in molecular weight; 90 kDa to 110 kDa in molecular weight; 90 kDa to 120 kDa in molecular weight; 90 kDa to 130 kDa in molecular weight; 90 kDa to 140 kDa in molecular weight; 90 kDa to 150 kDa in molecular weight; 90 kDa to 160 kDa in molecular weight; 100 kDa to 120 kDa in molecular weight; 100 kDa to 130 kDa in molecular weight; 100 kDa to 140 kDa in molecular weight; 100 kDa to 150 kDa in molecular weight; 100 kDa to 160 kDa in molecular weight; and similar desired molecular weight ranges. Any whole number integer within any of the above ranges is contemplated as an embodiment of the invention.

[0090] In one embodiment, the conjugate has a molecular weight of between about 50 kDa and about 5000 kDa in molecular weight. In one embodiment, the conjugate has a molecular weight of between about 200 kDa and about 5000 kDa in molecular weight. In one embodiment, the immunogenic conjugate has a molecular weight of between about 500 kDa and about 2500 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of between about 500 kDa and about 2500 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of between about 600 kDa and about 2800 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of between about 700 kDa and about 2700 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of between about 1000 kDa and about 2000 kDa; between about 1800 kDa and about 2500 kDa; between about 1100 kDa and about 2200 kDa; between about 1900 kDa and about 2700 kDa; between about 1200 kDa and about 2400 kDa; between about 1700 kDa and about 2600 kDa; between about 1300 kDa and about 2600 kDa; between about 1600 kDa and about 3000 kDa. Any whole number integer within any of the above ranges is contemplated as an embodiment of the vaccine composition as disclosed herein.

[0091] In one embodiment, the serotype 5 or 8 capsular polysaccharide has a degree of O-acetylation between 10-100%. In one embodiment, the degree of O-acetylation is between 50-100%. In one embodiment, the degree of O-acetylation is between 75-100%.

[0092] In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein can comprise additional polymers, for example, polyethylene glycol-based polymers, poly(ortho ester) polymers, polyacryl carriers, PLGA, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, β-amino ester polymers, polyphosphoester (PPE), liposomes, polymerosomes, nucleic acids, phosphorothioated oligonucleotides, chitosan, silk, polymeric micelles, protein polymers, virus particles, virus-like-particles (VLPs) or other micro-particles. See, e.g., El-Sayed et al., Smart Polymer Carriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5 Exp. Op. Biol. Therapy, 23 (2005). Biocompatible polymers developed for nucleic acid delivery may be adapted for 22 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT use as a backbone herein. See, e.g., BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., eds., John Wiley & Sons, Inc. Hoboken, NJ, 2011).

[0093] For example, VLPs resemble viruses, but are non-infectious because they do not contain any viral genetic material. The expression, including recombinant expression, of viral structural proteins, such as envelope or capsid components, can result in the self-assembly of VLPs. VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g., adeno- associated virus), Retroviridae (e.g., HIV), and Flaviviridae (e.g., Hepatitis B or C viruses). VLPs can be produced in a variety of cell culture systems including mammalian cell lines, insect cell lines, yeast, and plant cells. Recombinant VLPs are particularly advantageous because the viral component can be fused to recombinant antigens as described herein.

[0094] In some embodiments, a polysaccharide for use a vaccine composition as disclosed herein is part of a polysaccharide-protein conjugate. In some embodiments, a SBD protein as disclosed herein can be combined with the vaccine comprising a S. pneumonia polysaccharide-protein conjugate, for example PREVENAR 13 (alternative tradenames: PCV13, Prevnar 20, Prevnar 13, Synflorix, others; discontinued Prevnar (PCV7)) is a tridecavalent vaccine because it contains thirteen serotypes of pneumococcus (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F).

[0095] In some embodiments, a SBD protein as disclosed herein can be combined with the vaccine comprising a Neisseria meningitidis polysaccharide or a Neisseria meningitidis polysaccharide-protein conjugate, for example, but not limited to Menactra, Menveo, Menomune, Menquadfi, Nimenrix and Mencevax. The three conjugate vaccines (MCV-4), Menactra, Menveo and MenQuadfi, and the pure polysaccharide vaccine Menomune (MPSV4), Menveo and MenQuadfi are approved for medical use in the European Unio others; and targeting serogroups A, C, W-135, and Y Neisseria meningitidis.

[0096] In some embodiments, a SBD protein as disclosed herein can be combined with the vaccine INFANRIX HEXA (tradenames: ActHIB, Hiberix, OmniHIB), which is a Haemophilus influenzae type B vaccine, also known as Hib vaccine, is a vaccine used to prevent Haemophilus influenzae type b (Hib) infection.

[0097] In some embodiments, a polysaccharide for use a vaccine composition as disclosed herein is part of a polysaccharide-protein conjugate, were the protein non-covalently associates with the polysaccharide. In some embodiments, the vaccine composition a disclosed herein can comprise a SBD protein as disclosed herein and further comprise one or more Multiple presenting antigen systems (MAPS), such as those disclosed in in International Applications: WO / 2020 / 056127, WO / 2020 / 056202, WO / 2014 / 124228, WO / 2023 / 039223, US11560410B2, WO / 2018 / 183475, WO / 2018 / 217564, WO / 2023 / 102359, WO / 2023 / 102359A9, WO / 2012 / 155007, WO / 2023 / 192997A2WO / 2013 / 134656, WO / 2023 / 039108, WO / 2012 / 155053, WO / 2023 / 172741A2, WO / 2014 / 124228, WO / 2020 / 056127, WO / 2017 / 192801, WO / 2020 / 056202, WO / 2018 / 237221, WO / 2023 / 039223, US11305001, US20220362367, US20210008192, US20200121777, US20140154287, US10766932, US20210332090, US20230233667, US11560410, US10611805, US20160090404, US10017548, 23 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT US9499593, US20140154286, US20200407404, US20190119335, US20150374811, US11576958, US20230081705, US20230089151, US20200087361, US20190119332, US11013793, US11701416, US20210346487, US20200222522, US11612647, US20220072118, US20230091255, or US20150374811, or WO2023 / 102359, each of which is incorporated herein in its entirety by reference, or International applications: PCT / 2023 / 76822 and PCT / 2023 / 76878, and PCT / US2023 / 76909, filed on October 14, 2023, each of which is incorporated herein in its entirety by reference. (c) Polysaccharides from specific pathogens

[0098] As disclosed herein, the vaccine composition can comprise antigenic polysaccharides that comprise sialic acid. It is envisioned that polysaccharide from a pathogen can be used in a vaccine composition as disclosed herein, which are disclosed in Ghosh, S. (2020). Sialic acids and sialoglycoconjugates in the biology of life, health and disease. Academic Press, , and the reference of which is incorporated herein in its entirety. Polysaccharides from the following pathogens shown in Table 2A and Table 2B can be used in the vaccine composition as disclosed herein:

[0099] Table 2A:

[0100] Table 2B:24 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT(i) S. pneumoniae polysaccharides

[0101] In some embodiments an immunogenic polysaccharide for use in the vaccine composition as disclosed herein can be a pneumococcal polysaccharide, e.g., a capsular polysaccharide from Streptococcus pneumoniae from any of the over 93 serotypes of pneumococcus that have been identified to date, for example, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Additional pneumococcal serotypes may be identified and included in the present vaccine composition as described herein. More than one pneumococcal polysaccharide can be included as the polysaccharide in a vaccine composition as disclosed herein. In some embodiments, the vaccine composition comprises a pneumococcal polysaccharide from one serotype, and another pneumococcal polysaccharide from a different serotype. In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein is Type 1 capsular polysaccharide (CP1) from streptococcus pneumoniae.

[0102] In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein comprises a polysaccharide of Streptococcus pneumoniae having a serotype selected from one or more of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48, as disclosed in U.S. Patent 11,013,793, which is incorporated herein in its entirety by reference. In some embodiments, the immunogenic polysaccharide is a polysaccharide antigen of Streptococcus pneumoniae which comprises a polysaccharide of Streptococcus pneumoniae having a serotype selected from one or more of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20B, 22F, 23F, and 33F, as disclosed in U.S. Patent 11,013,793, which is incorporated herein in its entirety by reference.

[0103] In some embodiments, a vaccine as disclosed herein comprises a polysaccharide antigen from a distinct Streptococcus pneumoniae serotype, where the Streptococcus pneumoniae serotypes are selected from one or more of Streptococcus pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6F, 25 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11E, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33E, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F, and 48. (ii) S. Aureus polysaccharides

[0104] In some embodiments, a polysaccharide for use in a vaccine composition as disclosed herein is a polysaccharide or oligosaccharide from Gram-positive bacteria, for example, a Staphlococcus aureus capsular polysaccharide.

[0105] Type 5 and Type 8 Polysaccharides from S. aureus

[0106] Most strains of S. aureus that cause infection in man contain either Type 5 or Type 8 polysaccharides. Approximately 60% of human strains are Type 8 and approximately 30% are Type 5. The structures of Type 5 and Type 8 capsular polysaccharide antigens are described in Moreau et al Carbohydrate Res.201; 285 (1990) and Fournier et al Infect. Immun.45; 87 (1984). Both have FucNAcp in their repeat unit as well as ManNAcA which can be used to introduce a sulfhydryl group.

[0107] Recently (Jones Carbohydrate Research 340, 1097-1106 (2005)) NMR spectroscopy revised the structures of the capsular polysaccharides to:

[0108] Type 5→4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-

[0109] Polysaccharides may be extracted from the appropriate strain of S. aureus using methods well known to the skilled man, for instance as described in U.S. Pat. No.6,294,177 or Infection and Immunity (1990) 58(7); 2367, Fournier et al. (1984), supra; Fournier et al. (1987) Ann. Inst. Pasteur / Microbiol.138:561-567; US Patent Application Publication No.2007 / 0141077; and Int'l Patent Application Publication No. WO 00 / 56357; each of which is incorporated herein by reference as if set forth in its entirety). For example, ATCC 12902 is a Type 5 S. aureus strain and ATCC 12605 is a Type 8 S. aureus strain. In addition, they can be produced using synthetic protocols. Moreover, serotype 5 or 8 capsular polysaccharide can be recombinant produced using genetic engineering procedures also known to one of ordinary skill in the art (see, Sau et al. (1997) Microbiology 143:2395-2405; and U.S. Pat. No.6,027,925; each of which is incorporated herein by reference as if set forth in its entirety).

[0110] One S. aureus strain that can be used to obtain isolated serotype 8 capsular polysaccharide is S. aureus R2 PFESA0286. This strain was selected by flow cytometry with rabbit anti-serotype 8 polysaccharide antibodies after cultivation of S. aureus PFESA0286 (American Type Culture Collection; Manassas, Va.: ATCC Accession No.495:25) in Modified Frantz Broth. Two populations, R1 and R2, were observed during flow cytometry. R1 and R2 were purified and re-cultured. R2 yielded a serotype 8 capsular polysaccharide. Flow cytometric analysis showed a homogenous fluorescence 26 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT intensity. As such, R2 was selected for serotype 8 capsular polysaccharide production. One S. aureus strain that can be used to obtain isolated serotype 5 capsular polysaccharide is S. aureus PFESA0266. This strain produces serotype 5 capsular polysaccharide during growth, and production peaks when cells are in a stationary phase. Other S. aureus type 5 or type 8 strains can be used to make the respective polysaccharides that are obtained either from established culture collections or clinical specimens. In some embodiments, a Becker or Newman S. aureus strain can be used to obtain isolated serotype 5 capsular polysaccharide (CP5). In some embodiments, the Newman S. aureus strain can be used to obtain isolated serotype 5 capsular polysaccharide (CP5). In some embodiments, a Becker or Newman S. aureus strain can be used to obtain isolated serotype 8 capsular polysaccharide (CP8). In some embodiments, the Becker S. aureus strain can be used to obtain isolated serotype 8 capsular polysaccharide (CP8).

[0111] Polysaccharides are of native size or alternatively may be sized, for instance by microfluidisation, ultrasonic irradiation or by chemical treatment. The invention also covers oligosaccharides derived from the type 5 and 8 polysaccharides from S. aureus.

[0112] In some embodiments, an polysaccharide for use in a vaccine composition as disclosed herein can comprises a Type 5 (CP5), or Type 8 (CP8) capsular polysaccharides (CP), or any of the polysaccharides or oligosaccharides or lipopolysaccharides from Staphylococcus aureus. In some embodiments, an polysaccharide for use in a vaccine composition as disclosed herein can comprises a capsular polysaccharide from a non-typeable (NT) SA strain, e.g., a cell wall surface antigen 336 (Type 336) or a polyribitol phosphate N-acetylglucosamine, which resembles cell wall teichoic acid. Type 336 isolates do not express capsule but do express cell surface polysaccharide or the 336 polysaccharide (336PS), which resembles S. aureus cell wall teichoic acid (Ma, J., et al., 2004. Evaluation of serotypes of Staphylococcus aureus strains used in the production of a bovine mastitis bacterin. J. Dairy. Sci. 87:178-18214, 17; O'Brien, et al., 2000. Production of antibodies to Staphylococcus aureus serotypes 5, 8, and 336 using poly(dl-lactide-co-glycolide) microspheres. J. Dairy Sci.83:1758-1766).

[0113] In some embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein can comprises a capsular polysaccharide (CP) from a methicillin-resistant S. aureus (MRSA), including hospital-acquired MRSA (HA-MRSA), or community-acquired MRSA (CA-MRSA) or any polysaccharides or oligosaccharides or lipopolysaccharides from MRSA, e.g., e.g., any one or more of a CP5, or CP8 from HA-MSSA and / or CA-MRSA. In alternative embodiments, an immunogenic polysaccharide for use in the vaccine composition as disclosed herein can comprises a capsular polysaccharide (CP) from a methicillin-sensitive S. aureus (MSSA), e.g., any one or more of a CP5, or CP8 from MSSA.

[0114] The association of particular capsule serotypes with disease is possible through monitoring of clinical isolates. Of the eight different serotypes of S. aureus identified (Karakawa and Vann (1982) only serotypes 1 and 2 are heavily encapsulated, and these are rarely isolated. See Capsular Polysaccharides of Staphylococcus aureus, p.285-293, In J. B. Robbins, J. C. Hill and J. C. Sadoff 27 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT (ed.), Seminars in infectious disease, vol.4, Bacterial Vaccines. Thieme Stratton, Inc. New York). Surveys have shown that approximately 85-90% of S. aureus clinical isolates express CP5 or CP8 (Arbeit R D, et al., Diagn. Microbiol. Infect. Dis. (1984) April; 2(2):85-91; Karakawa W W, et al., J. Clin. Microbiol. (1985) September; 22(3):445-7; Essawi T, et al., Trop. Med. Int. Health. (1998) July; 3(7):576-83; Na'was T, et al., J. Clin. Microbiol. (1998) 36(2):414-20. Most of CP5 and CP8 non- typeable strains are genetically type 5 or type 8 containing mutations in cap5 / 8 locus (Cocchiaro, Gomez et al., (2006), Mol. Microbiol. February 59(3):948-960). Capsulation for some strains is lost rapidly within few passages in vitro which is due to a repressive effect of high phosphate concentration in media used in clinical diagnosis on capsule production. It was also reported that non-capsulated isolates recover capsule expression after passing through cows. See Opdebeck, J. P. et al., J. Med. Microbiol.19:275-278 (1985). Some non-typeable strains become capsule positive under appropriate growth conditions.

[0115] CP5 and CP8 Structure: The repeat unit of both CP5 and CP8 is comprised of 2-acetamido-2- deoxy-D-mannuronic acid, 2-acetamido-2-deoxy-L-fucose and 2-acetamido-2-deoxy-D-fucose. See C. Jones et al., Carbohydr. Res.340:1097-1106 (2005). Although CP5 and CP8 have the same sugar composition, they have been demonstrated to be immunologically distinct. They differ in glycosidic linkages and site of O-acetylation of uronic acid. Strain dependent incomplete N-acetylation of one of the FucNAc residues was observed. See Tzianabos et al., PNAS V98: 9365 (2001). (iii) GBS polysaccharides

[0116] In some embodiments, a polysaccharide in a vaccine composition as described herein includes one or more GBS polysaccharides (PS). In some embodiments, a vaccine composition as described herein comprises a polysaccharide from GBS. In some embodiments, a vaccine composition includes one or more GBS capsular polysaccharides or O-specific polysaccharides (OSP) from, or derived from, one or more GBS subtypes selected from group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX.

[0117] In some embodiments, a vaccine composition described herein comprises a GBS polysaccharide that is > 60kDa, or > 70kDa, or > 80kDa, or > 90kDa, or > 100kDa, or > 110kDa, or > 120kDa. In some embodiments, a vaccine composition as described herein comprises an OSP polysaccharide from GBS that is between 90-110kDa.

[0118] In some embodiments, a vaccine composition comprises a polysaccharide from Streptococcus agalactiae. The polysaccharide may be isolated from any encapsulated strain of S. agalactiae, such as 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB 4052 (ATCC Accession No.31574), H36B (ATCC Accession No.12401), S40, S42, MB 4053 (ATCC Accession No.31575), M709, 133, 7357, PFEGBST0267, MB 4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No.12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB 4082 (ATCC Accession No.31577), M132, 110, M781 28 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT (ATCC Accession No. BAA-22), D136C(3) (ATCC Accession No.12403), M782, S23, 120, MB 4316 (M-732; ATCC Accession No.31475), M132, K79, COH1 (ATCC Accession No. BAA-1176), PFEGBST0563, 3139 (ATCC Accession No.49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111(ATCC Accession No. BAA-23), CJB112, 2603 V / R (ATCC Accession No. BAA-611), NCTC 10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B 4589, B 4645, SS1214, CZ- PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-N1-016, IT-PW-62, and IT-PW-64. Polysaccharides isolated from Streptococcus agalactiae useful in the immunogenic complexes as disclosed herein are disclosed in US Patent 10,226,525, which is incorporated herein in its entirety by reference.

[0119] Methods of Isolating and Purifying Polysaccharides

[0120] In some embodiments, the disclosure provides methods of purifying one or more polysaccharides described herein from a pathogen or from cellular components of bacteria. In some embodiments, methods comprise purifying capsular polysaccharides from one or more cellular components of bacteria. 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.

[0121] 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.

[0122] Methods of Biotinylating Polysaccharides

[0123] 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 method comprises reacting purified polysaccharides with 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). In some embodiments, the biotinylated polysaccharides are purified by filtration to remove process residuals such as unreacted biotin, dimethylaminopyridine, acetonitrile, cyanide and unreacted glycine.

[0124] In some embodiments, a vaccine composition or vaccine as described herein, upon administration to a subject, induces an immune response against the pathogen from which the one or more antigenic polysaccharide originates from, including induces an immune response against one or 29 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT more serotypes of a pathogen that the antigenic polysaccharide originates from. The phrases as used herein “a pathogen that an antigenic polysaccharide originates from” or “a pathogen from which the antigenic polysaccharide was derived” can be used interchangeably, and refer to a pathogen that comprises the same type of polysaccharide that is used as an antigenic polysaccharide present in the immunogenic or vaccine composition.

[0125] In some embodiments, the immunogenic composition or vaccine, upon administration to a subject, induces a protective immune response against one or more serotypes of a pathogen that polysaccharide originates from. 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 Th1, Th2, or Th17 response, or a CD8+ T cell response, or a CD4+ and a 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.

[0126] In some embodiments, a vaccine composition described herein that includes one or more antigenic polysaccharides is characterized in that one or more of the opsonization potential, or immune response to one or more antigenic polysaccharides is increased relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, one or more of the opsonization potential, immune response to the one or more antigenic polysaccharides is increased at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a predetermined level, as measured by ELISA and or by a functional antibody assay. In some embodiments, the predetermined level is a pre-immune level. In some embodiments, the predetermined level is a pre-immune level. In some embodiments, one or more polypeptide antigens are carrier proteins for one or more antigenic polysaccharides.

[0127] In some embodiments, a vaccine composition 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 composition comprising an antigenic polysaccharide without the presence of a SBD protein as disclosed herein. In some embodiments, a vaccine composition as described herein, upon administration to a subject, induces a protective immune response.

[0128] The vaccine composition as described herein may be used for prophylactic and / or therapeutic treatment for a pathogen, where the antigenic polysaccharide is from of the pathogen. Accordingly, this application provides a method for immunizing a subject suffering from or susceptible to a pathogen infection, comprising administering an immunologically effective amount of a vaccine formulations described herein. The subject receiving the vaccination may be a male or a female, and may be an infant, child, adolescent, or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal. In some embodiments, an immunogenic complex 30 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT described herein, upon administration to a subject, induces a protective immune response against one or more serotypes of a pathogen from which the antigenic polysaccharide was derived.

[0129] In therapeutic embodiments, a vaccine composition as disclosed herein may be administered to a subject suffering from an infection or disease caused by a pathogen from which the antigenic polysaccharide was derived, in an amount sufficient to treat the subject. Treating the subject, in this case, can relate to reducing a symptom and / or bacterial load and / or sequelae in an infected subject. In some embodiments, treating the subject refers to reducing the duration of symptoms or sequelae, or reducing the intensity of symptoms or sequelae. In some embodiments, the vaccine reduces transmissibility of the pathogen and / or infection from the vaccinated subject. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0130] In therapeutic embodiments, a vaccine composition as disclosed herein is administered to a subject postinfection. 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.

[0131] In some embodiments, a vaccine compositions as disclosed herein 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%. In particular embodiments, vaccinated subjects may display no symptoms or sequelae upon contact with a pathogen from which the antigenic polysaccharide was derived, do not become colonized by such a pathogen, or both. 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 Th1 or Th17 cells.

[0132] In some embodiments, a vaccine composition as described herein, where upon administration to a subject, it induces an immune response against multiple serotypes of a pathogen from which the antigenic polysaccharide was derived. In some embodiments, a vaccine composition as disclosed herein, upon administration to a subject, induces an immune response, including but not limited to, a protective immune response, against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more) serotypes of a pathogen from which the antigenic polysaccharide was derived. 31 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0133] In some embodiments, the vaccine composition as disclosed herein induces a greater immune response to the antigenic polysaccharide than a composition of the same polysaccharide in the absence of a SBD protein. 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 Th1, 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.

[0134] 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 composition comprising an antigenic polysaccharide alone or in the absence of a SBD protein. 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 level greater than a composition comprising a polypeptide antigen alone, or in the absence of a SBD protein. In some embodiments, the immune response is a protective immune response.

[0135] In some embodiments, a vaccine composition as described herein, upon administration to a subject, induces an immune response against a pathogen in the subject at a level greater than a control composition, where the control composition is a vaccine with the same polysaccharide without a SBD protein. In some embodiments, the immunogenic composition or vaccine, upon administration to a subject, induces an immune response against one or more serotypes of a pathogen, where the pathogen comprises the same type of polysaccharide present in the vaccine composition, 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, where the control composition is a vaccine composition comprising the same polysaccharide in the absence of a SBD protein as disclosed herein.

[0136] In some embodiments, a vaccine composition as described herein, upon administration to a subject, induces an immune response that can help protect against the establishment of infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine protects against colonization at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine inhibits infection by a pathogen, e.g., a pathogen from which the antigenic 32 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT polysaccharide was derived, in a non-colonized or uninfected subject at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine reduces the duration of colonization by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, in a subject who is already colonized 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, where the control composition is a vaccine composition comprising the same polysaccharide in the absence of a SBD protein as disclosed herein. X. Vaccine Formulations

[0137] In some embodiments, a vaccine composition is a polyvalent or multivalent vaccine. In some embodiments, the valency of a vaccine composition refers to the number of different polysaccharide species 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. In a non-limiting example, a 6-valent (6V) vaccine composition may comprise more than 6 antigenic components (e.g., peptide and / or polysaccharide components) and may induce an immunoprotective response against more than 6 pathogens, or pathogenic serotypes or strains.

[0138] In some embodiments, the combined weight of polysaccharides and SBD polypeptide in the vaccine composition is about 0.20 ug. In some embodiments, the combined weight of polysaccharides and SBD protein in the vaccine composition is about 0.40 ug. In some embodiments, the combined weight of polysaccharides and SBD protein in the vaccine composition is about 1 ug. In some embodiments, the combined weight of polysaccharides and SBD protein in the vaccine composition is about 2 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine contributed by each immunogenic composition is about 3 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 4 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 5 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 6 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 7 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 8 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 9 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is 33 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT about 10 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 11 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 12 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 14 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 16 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 18 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 20 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 21 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 22 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 23 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 24 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 25 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 30 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 40 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 50 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 60 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 70 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 80 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 90 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 100 ug. In some embodiments, the combined weight of polysaccharides and polypeptides in the vaccine composition is about 110 ug.

[0139] The vaccine composition as 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. 34 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0140] In some embodiments, a vaccine composition as described herein 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. Preparation of pneumococcal polysaccharide and conjugate vaccines is described, for example, in USSN 11 / 395,593, filed March 31, 2006, the contents of which are incorporated herein by reference.

[0141] In general, pharmaceutically acceptable carrier(s) include solvents, dispersion media, and the like, which are compatible with pharmaceutical administration. For example, materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose, dextrose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; polyols such as glycerol, propylene glycol, and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator (Martin, 1975).

[0142] Vaccines may be formulated by combining one or more fusion proteins described herein with carriers and / or other optional components by any available means including, for example, conventional mixing, granulating, dissolving, lyophilizing, or similar processes.

[0143] Vaccines comprising one or more fusion proteins described herein 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.

[0144] 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, 35 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT then retaining this temperature for 6 h, then gradually adjusting to +34°C over 3 h, and finally retaining this temperature over 9 h.

[0145] In some embodiments, a vaccine composition as described herein 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.

[0146] Vaccines or vaccine components for use in accordance with the present disclosure may be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or immune-stimulating complexes (ISCOMS).

[0147] In certain situations, it may be desirable to prolong the effect or release of a vaccine 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 polymer 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.

[0148] Examples of biodegradable polymers that can be employed in accordance with the present disclosure include, for example, poly(orthoesters) and poly(anhydrides). One particular exemplary polymer is polylactide-polyglycolide.

[0149] Depot injectable formulations may also be prepared by entrapping the product in liposomes or microemulsions, which are compatible with body tissues.

[0150] 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).

[0151] Vaccines comprising one or more fusion proteins described herein for use in accordance with the present disclosure include immunogenic compositions, and may additionally include one or more 36 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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. (a) Adjuvants

[0152] In some embodiments, a vaccine composition as 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 Cug-containing DNA, which activate cells of the innate immune system.

[0153] 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).

[0154] Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the generation, proliferation, or activation of Th1 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.

[0155] In some 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 Th17 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 37 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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 Th17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL-17 may be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli, or ETEC), activate a Th17 response. Thus, in some embodiments, the adjuvant is a toxin or toxoid. Cholera toxin was successfully used in a mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1. One form of labile toxin is produced by Intercell. 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).

[0156] 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 some 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.

[0157] Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a Th17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram-negative bacteria, such as S. minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans et al, 2003. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka et al, 2007). Various TLR agonists (including TLR-4 agonists) have been produced and / or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR- activating adjuvant is R848 (resiquimod). 38 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0158] 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.

[0159] In some embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.

[0160] In some embodiments, the vaccine composition as disclosed herein comprises an AddaSO3 adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises a R848 adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises an ODN2395 adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises an Alum phos adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises an AddaSO3 alum adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises a R848 alum adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises an ODN alum adjuvant. In some embodiments, the vaccine composition as disclosed herein comprises an Alum 2PE adjuvant.

[0161] In some embodiments, the pharmaceutical composition as disclosed herein comprises an AddaSO3 adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises a R848 adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises an ODN2395 adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises an Alum phos adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises an AddaSO3 alum adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises a R848 alum adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises an ODN alum adjuvant. In some embodiments, the pharmaceutical composition as disclosed herein comprises an Alum 2PE adjuvant.

[0162] In some embodiments, the immunogenic composition as disclosed herein comprises an AddaSO3 adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises a R848 adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises an ODN2395 adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises an Alum phos adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises an AddaSO3 alum adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises a R848 alum adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises an ODN alum adjuvant. In some embodiments, the immunogenic composition as disclosed herein comprises an Alum 2PE adjuvant.

[0163] In some embodiments, the adjuvant can comprise of at least one of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, at least two of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, 39 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT at least three of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, at least four of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, at least five of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, at least six of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, at least seven of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE, or at least eight of AddaSO3, R848, ODN2395, Alum phosphate, AddaSO3 alum, R848 alum, ODN alum, or Alum 2PE.

[0164] 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).

[0165] In some embodiments, vaccine composition as described herein is 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.

[0166] 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.

[0167] Vaccines for use in accordance with the present disclosure may include, or be administered concurrently with, antimicrobial therapy. 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, penicillin, vancomycin, erythromycin, azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.

[0168] 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. (b) Additional Components and Excipients

[0169] In addition to the fusion proteins described herein and the adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components. 40 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0170] In some embodiments, the vaccine formulation comprises aluminum phosphate (referred to herein as alum phosphate, or AP). In some embodiments, a vaccine formulation comprising S. Paratyphi-MAPS aluminum phosphate (referred to herein as alum phosphate, or AP). In some embodiments, the amount of alum phosphate is determined by one of ordinary skill in the art. In some embodiments, the amount of alum phosphate is 250µg per 500µl injection (25µg polysaccharide). In some embodiments, a vaccine formulation or immunogenic composition comprises 250µg of alum phosphate per 500µl injection. In some embodiments, the alum phosphate is in a buffer comprising 20mM Histadine, pH 6, 150 mM NaCl, 0.02% tween 80.

[0171] In some 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.

[0172] In some 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.

[0173] In some 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-(1,1,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100), and 4- (1,1,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or nonionic.

[0174] In some embodiments, the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

[0175] In some 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 multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In some embodiments, the preservative is 2- phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and / or sorbic acid.

[0176] In another aspect of the invention, the vaccine composition is lyophilized, optionally in the presence of at least one excipient. In one embodiment, the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCI), dried skim milk, glycerol, propylene glycol, water, and ethanol. In a preferred embodiment, the at least one excipient is selected from the group consisting of sucrose, mannitol, and glycine. In a particular embodiment, the at least one 41 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT excipient is sucrose.. In one aspect, the lyophilized composition comprises about 1 % (w / v) to about 10% (w / v) of the at least one excipient, preferably greater than about 5.5% (w / v). In another embodiment, the lyophilized composition comprises an additional excipient. In one such embodiment, the additional excipient is mannitol or glycine. In a preferred embodiment, the lyophilized composition comprises about 1 % (w / v) to about 10% (w / v) of the additional excipient. In yet another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), an adjuvant suspension, or saline. In a particular embodiment, the diluent is a suspension of any adjuvant described herein, such as an aluminum-based adjuvant suspension, preferably an aluminum phosphate suspension. XI. Methods of Administration

[0177] In some embodiments, a vaccine composition as described herein is administered to a subject at risk of developing an infection or disease from a pathogen, e.g. an infant, a toddler, a juvenile, or an older adult. In some embodiments, the immunogenic composition or vaccine is administered to a subject at elevated risk of developing or being infected with a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, e.g., immunocompromised subjects, subjects having sickle cell disease or other hemoglobinopathies, congenital or acquired asplenia, splenic dysfunction, chronic renal failure or nephrotic syndrome, diseases associated with treatment with immunosuppressive drugs or radiation therapy, including malignant neoplasm, leukemia, lymphomas, Hodgkin's disease, or solid organ transplantation, congenital or acquired immunodeficiency, HIV infection, cerebrospinal fluid leaks, cochlear implant(s), chronic heart disease, chronic lung disease, diabetes mellitus, alcoholism, chronic liver disease, cigarette smoking, asthma, generalized malignancy, multiple myeloma, or solid organ transplantation. It will be appreciated that a subject can be considered at risk for developing a disease without having been diagnosed with any symptoms of the disease. For example, if the subject is known to have been, or to be intended to be, in situations with relatively high risk of infection, that subject will be considered at risk for developing the disease.

[0178] Any effective route of administration 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, the immunogenic composition or vaccine 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). Intranasal administration may be particularly useful in some contexts. In some embodiments, it may be desirable to administer different doses of the immunogenic composition or vaccine by different routes; in some embodiments, it may be desirable to administer different components of one dose via different routes.

[0179] In some embodiments, pharmaceutical compositions (e.g., immunogenic compositions or 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 42 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0180] 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 immunogenic compositions or vaccines 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).

[0181] As described above, pharmaceutical compositions (e.g., immunogenic compositions or 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 or vaccines, within a period of less than 24 h, is considered a single dose. To give but one example, immunogenic compositions or vaccines having different antigenic components may be administered in separate compositions, but as part of a single dose. As noted above, such separate compositions may be administered via different routes or via the same route. Alternatively or additionally, in embodiments wherein an immunogenic composition or vaccine is combined with additional types of active agents, the immunogenic composition or vaccine may be administered via one route, and a second active agent may be administered by the same route or by a different route.

[0182] Pharmaceutical compositions (e.g., immunogenic compositions or vaccines) are administered in such amounts and for such time as is necessary to achieve a desired result. In some embodiments of the present invention, the immunogenic composition or vaccine 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.

[0183] The amount of a SBD and polysaccharide as described herein in each pharmaceutical composition (e.g., immunogenic composition or vaccine) dose is selected to allow the vaccine, when 43 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT administered as described herein, to induce an appropriate immunoprotective response without significant adverse side effects.

[0184] In some embodiments, a pharmaceutical composition as described herein induces a Th1 and / or Th17 cell response upon administration to a subject. In some embodiments, the pharmaceutical composition induces an opsonic / bactericidal response against a pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived, upon administration to a subject. In some embodiments, a vaccine composition as disclosed herein reduces rate of transmission and / or colonization of the mucosal surfaces by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, upon administration to a subject. In some embodiments, the pharmaceutical composition reduces rate of transmission and / or colonization of the nasopharynx or the lungs by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, upon transmission.

[0185] Some embodiments provide for a method of immunizing a subject against infection of a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, comprising administering to the subject a vaccine composition as described herein. Some embodiments provide for a method of immunizing a subject against infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, comprising administering to the subject an immunologically effective amount of a vaccine composition as described herein. Some embodiments provide for a method of immunizing a subject against infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived, comprising administering to the subject an immunologically effective amount of a pharmaceutical composition comprising a fusion protein described herein. (a) Combination Prophylaxis or Combination Therapy

[0186] In some embodiments, a vaccine composition as described herein may be administered in combination with another agent. In some embodiments, the agent is or comprises PCV13. In some embodiments, the agent is or comprises PPSV23. In some embodiments, the agent is or comprises an antibiotic. (b) Dosing

[0187] In some embodiments, administration of a vaccine composition as 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 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.

[0188] 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. Such 44 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT additional immunization doses can be referred to as boosters. In some embodiments, a booster (or second or subsequent) immunization dose is administered 2 weeks, or 3 weeks, or about 1 month, or about 2 months, or about 6 months or about 1 year after the preceding dose (where the proceeding dose can be initial dose or a second or third dose, or booster dose).

[0189] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to an infant subject. In some embodiments, the infant subject is 18 months old or younger. In some embodiments, the infant subject is 12 months old or younger. In some embodiments, the infant subject has previously received one or more doses of a vaccine composition as disclosed herein; in other embodiments, the infant subject is naïve to a polysaccharide vaccine. In some embodiments, the infant subject has previously been infected with, or exposed to infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived.

[0190] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to a toddler subject. In some embodiments, the toddler subject is 5 years old or younger. In some embodiments, the toddler subject is 4 years old or younger. In some embodiments, the toddler subject has previously received one or more doses of a polysaccharide vaccine; in other embodiments, the toddler subject is naïve to vaccines. In some embodiments, the toddler subject has previously been infected with, or exposed to infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived.

[0191] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to a juvenile subject. In some embodiments, the juvenile subject is 18 years old or younger. In some embodiments, the juvenile subject is 15 years old or younger. In some embodiments, the juvenile subject has previously received one or more doses of a polysaccharide vaccine; in other embodiments, the juvenile subject is naïve to vaccines. In some embodiments, the juvenile subject has previously been infected with, or exposed to infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived.

[0192] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to an adult subject. In some embodiments, the adult subject is older than about 50 years of age. In some embodiments, the adult subject is older than about 65 years of age. In some embodiments, the adult subject has previously received one or more doses of a polysaccharide vaccine; in other embodiments, the adult subject is naïve to vaccines. In some embodiments, the adult subject has previously been infected with, or exposed to infection by a pathogen, e.g., a pathogen from which the antigenic polysaccharide was derived.

[0193] 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 45 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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 or vaccines disclosed herein, found in the standard schedule, or for the administration of immunogenic compositions or 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 immunization schedules are provided for illustrative purposes. Detailed descriptions of methods to assess immunogenic response discussed herein allow one to develop alterations to the sample immunization schedules without undue experimentation.

[0194] In one embodiment of the present disclosure, a first administration of a vaccine composition as disclosed herein usually 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.

[0195] In one embodiment of the present disclosure, a first administration of a vaccine composition as disclosed herein usually occurs when a subject is 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.

[0196] 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 polypeptides, polysaccharide(s) and / or conjugate(s) or combinations thereof is / are strong, and in such a situation a single dose schedule is sufficient to induce a lasting immune-protective response.

[0197] In some 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.

[0198] Without being bound by theory, a first dose of vaccine administered according to the disclosure may be considered a “priming” dose. In some embodiments, more than one dose is included in an immunization schedule. In such a scenario, a subsequent dose may be considered a “boosting” dose.

[0199] A priming dose may be administered to a naïve subject (a subject who has never previously received a conjugated polysaccharide vaccine). In some embodiments, a priming dose may be administered to a subject who has previously received conjugated polysaccharide 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 conjugated polysaccharide vaccine at least twenty or more years previous to administration of a priming vaccine according to the invention.

[0200] 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 46 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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 some 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 some embodiments, intervals between subsequent doses may decrease as the subject ages.

[0201] 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. (c) Assays for Determining Immune Response

[0202] In some embodiments, a method of assessing the immunogenicity of a pharmaceutical composition, e.g., a vaccine composition as disclosed 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, serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of infections by a pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived. Animal models of infection for S. pneumococcus and Streptococcus agalactiae disease are well known (e.g. pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization). Parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with a pathogen of interest that are the targets of the immunogenic composition, e.g., a pathogen from which the antigenic polysaccharide was derived. In some embodiments, the immune response is compared to a control composition, including but not limited to vaccines comprising polysaccharides of the same pathogen where a SBD protein is not present.

[0203] In some embodiments, a method of assessing the potency of a pharmaceutical composition, e.g., a vaccine composition as disclosed herein comprises evaluating, measuring, and / or comparing an 47 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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, serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of the pathogen which is the target of the vaccine. Animal models of infection for S. pneumococcus and Streptococcus agalactiae disease are well known (e.g. pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization). Parameters include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with a pathogen of interest that is the target of the immunogenic composition, e.g., a pathogen from which the antigenic polysaccharide was derived. In some embodiments, the immune response is compared to a control composition.

[0204] 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, IgG1, IgG2, IgM, IgA, etc.) to specific pathogen antigens (e.g., polypeptides or polysaccharides, 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 antigens. 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 antigens.

[0205] 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).

[0206] In the ELISA detection method, specific antibodies from the sera of vaccinated subjects are quantitated by incubation with antigens (e.g., polypeptides or polysaccharides, 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 IgG1 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. Antigens are conjugated to spectrally distinct microspheres that are mixed and incubated with serum. The antibodies bound to the antigens on the 48 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT coated microspheres are detected using a secondary antibody (e.g., R-Phycoerythrin-conjugated goat anti-human IgG).

[0207] An approach for assessing functional antibody in serum is the opsonophagocytic assay (OPA) which quantitates only the antibodies that can opsonize the bacteria, leading to ingestion and killing of the bacteria. The standard assay utilizes a human phagocytic effector cell, a source of complement, bacteria, and diluted sera. The assay readout is the serum endpoint titer at which there is >50% killing compared to bacteria incubated with complement and human cells alone (Romero-Steiner et al, 1997). This killing OPA can also be multiplexed by utilizing target strains of pathogen that carry different antibiotic resistance markers (Kim et al, 2003). Another type of multiplex opsonic assay is a nonkilling assay in which the uptake by phagocytic effector cells of fluorescent stained encapsulated pathogen or fluorescent microspheres conjugated with antigens from a target pathogen in the presence of diluted sera plus a complement source is evaluated by FC (Martinez et al, 1999). Opsonic activity of serum antibody plus complement can also be evaluated by measuring the oxidative response of phagocytic human effector cells to ingested pathogen (Munro et al.1985; Ojo-Amaize et al.1995).

[0208] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by immunogenic compositions or vaccines comprising a fusion protein described herein. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which provides protection against pneumonia, bacteremia, colonization of organs or tissues, or mortality is determined (Stack et al.1998; Saeland et al.2000).

[0209] In some embodiments, efficacy of immunization may be determined by assaying one or more cytokine levels by stimulating T cells from a subject after immunization. The one or more cytokine levels may be compared to the one or more cytokine levels in the same subject before immunization. Increased levels of the one or more cytokine, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase over pre-immunization cytokine levels, would indicate an increased response to the immunogenic composition or vaccine. In some embodiments, the one or more cytokines are selected from GM-CSP; IL-1α; IL-1β; 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-α; IFN-β; IFN-γ; MIP-1α; MIP- 1β; TGF-β; TNFα, or TNF-β. In a non-limiting example, efficacy of immunization may be determined by assaying IL-17 levels (particularly IL-17A) by stimulating T cells from a subject after immunization. The IL-17 levels may be compared to IL-17 levels in the same subject before immunization. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the immunogenic composition or vaccine.

[0210] In some embodiments, one may assay neutrophils in the presence of T cells or antibodies from the patient for pathogenic killing. Increased killing by the pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived, 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 composition. For example, one may measure Th17 cell activation, where increased Th17 cell activation, such as a 1.5 49 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the immunogenic composition or vaccine. In another non-limiting example, one may measure Th1 cell activation, where increased Th1 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 composition. One may also measure levels of an antibody specific to the immunogenic composition or 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 efficacy. In some embodiments, two or more of these assays are used. For example, one may measure IL-17 levels and the levels of immunogenic composition- or vaccine-specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of infection with the pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived, in vaccinated individuals compared to unvaccinated individuals.

[0211] Immunogenic composition or 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 composition to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of the pathogen of interest. In some cases, a challenge dose administered intranasally is sufficient to cause colonization (especially nasal colonization) by the pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived, in an unvaccinated animal, and in some cases a challenge dose administered via aspiration is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intraperitoneal injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intravenous injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals.

[0212] 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).

[0213] Parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with the pneumococcal pathogens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise an antigenic polysaccharide present in the immunogenic composition and not comprise an antigenic polypeptide present in the immunogenic composition. In some embodiments, a control composition may comprise an antigenic polypeptide present in the 50 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT immunogenic composition and not comprise an antigenic polysaccharide present in the immunogenic composition. In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition, and not comprise an antigenic polysaccharide and / or an immunogenic polypeptide present in the immunogenic composition.

[0214] In some embodiments, a method of assessing the potency of an immunogenic composition described herein comprises evaluating, measuring, and / or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, Th1 / Th17 cell response, cytokine level measurement and functional antibody levels as measured by OPK, serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of a pathogen of interest, for example, if the pathogen is a S. pneumococcus, an animal model of pneumococcal disease (e.g. pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization) can be used. Parameters of in vivo assays include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with the pneumococcal pathogens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise an antigenic polypeptide present in the immunogenic composition and not comprise an antigenic polysaccharide present in the immunogenic composition. In some embodiments, a control composition may comprise an adjuvant present in the immunogenic composition, and not comprise an antigenic polysaccharide and / or a SBD protein in the a vaccine composition as disclosed herein.

[0215] In some embodiments, a method of assessing a vaccine composition as disclosed 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, serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of a pathogen of interest, e.g., a pathogen from which the antigenic polysaccharide was derived. In some embodiments, an animal disease model is based on a model of pneumococcal disease (e.g. pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization). Parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with the pneumococcal pathogens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition. In some embodiments, a control composition may comprise an 51 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT antigenic polysaccharide present in the vaccine composition but not comprise a SBD protein as disclosed herein. X. Manufacture of Immunogenic Complexes

[0216] In some embodiments, SBD proteins as described herein may be non-covalently associated with antigenic polysaccharides as disclosed herein, e.g., by SBD interaction with sialic acid on the antigenic polysaccharide or van de Waals interactions with positively charged polysaccharides, as disclosed herein. In some embodiments, the polysaccharide is a purified antigenic polysaccharide, including a purified lipidated capsular polysaccharide oligosaccharide.

[0217] In some embodiments, a SBD protein as described herein is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioavailability, or immunogenicity of the fusion protein. Molecules that may be covalently bound to the fusion protein include a carbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, ranging from molecular weights of below 300 g / mol to over 10,000,000 g / mol. PEG chains can be linear, branched, or with comb or star geometries. In some embodiments, the fusion protein is covalently bound to a moeity that stimulates the immune system. An example of such a moeity is a lipid moeity. In some instances, lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innate immune system.

[0218] In some embodiments, a SBD protein and one or more additional components described herein are mixed together using known methods to form a multi-component immunogenic composition. In some embodiments, a SBD protein and one or more additional components described herein are nano- encapsulated using known methods. In some embodiments, a SBD and one or more additional components described herein are molded into nano- or micro- particles using known methods.

[0219] In some embodiments, the average (e.g., the mean) SBD protein to polysaccharide ratio in a vaccine composition disclosed herein 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:PS ratios are chosen to enhance the polysaccharide immunogenicity potential and / or to elicit protection against, or to inhibit, colonization by the pathogen of interest (independent of polysaccharide serotype) through a protein-specific immune response. In some embodiments, a vaccine composition as disclosed herein may comprise mixtures of different polysaccharides with different average protein to polysaccharide ratios.

[0220] In some embodiments, a vaccine composition comprises a different antigenic polysaccharides comprising a plurality of any one, or more distinct types of SBD proteins as disclosed herein. In some embodiments, the average ratio of a SBD protein as disclosed herein to a antigenic polysaccharide from a pathogen of interest 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 52 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT of total protein of a SBD protein as disclosed herein, to a polysaccharide from or derived from any pathogen of interest 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 total protein selected from any one or more of, or a combination of: SBD1, SBD2, SBD3, SBD4, NanH, NanH2, NanH3,VcNanH as disclosed herein, to a polysaccharide from or derived from any pathogen of interest 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]).

[0221] In some embodiments, the average ratio of total amount of SBD protein as disclosed herein to a polysaccharide from or derived from a pathogen of interest is chosen to enhance the polysaccharide immunogenicity potential and / or to elicit protection against, or to inhibit, pathogenic colonization by any subtype of the pathogen (independent of polysaccharide serotype) through a protein specific immune response. Immunogenic compositions and vaccines of the invention may comprise mixtures of different average protein to polysaccharide ratios. XII. Certain Definitions

[0222] 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.

[0223] 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.

[0224] 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., 53 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0225] Agent: In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0226] Amino acid: In its broadest sense, the term “amino acid”, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N– C(H)(R)–COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D- amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Non- standard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more 54 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0227] 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: CH1, 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 55 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in some 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, in some 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 IgNARor fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small ModularImmunoPharmaceuticals (“SMIPsTM”); 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 payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]).

[0228] Antigen: The term “antigen”, as used herein, refers to (i) an agent that induces an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen induces a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen induces a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen induces a humoral response and a cellular response. In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer (e.g., other than a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a 56 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0229] 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.

[0230] 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).

[0231] Carrier protein: As used herein, the term “carrier protein” refers to a protein or peptide that is coupled, complexed, or otherwise associated with a hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g., a polysaccharide) and that induces or improves an immune response to such a coupled, or complexed, or otherwise associated hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g., a polysaccharide). In some embodiments, such an immune response is or comprises a response to a hapten or less immunogenic antigen that is coupled, complexed, or otherwise associated with such a carrier protein. In some embodiments, such an immune response is or comprises a response to both a carrier protein and a hapten or less immunogenic antigen that is coupled, 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 such embodiments, immune response to such a carrier protein is 57 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT strong. In some embodiments, a carrier protein is coupled, complexed, or otherwise associated with one or more other molecules.

[0232] Colonization: As used herein, the term “colonization” generally refers to the ability of a microbe to grow at a target site or surface. For example, the term “colonization” refers to the ability of a microbe (e.g., a bacterium) to grow at an anatomical site (e.g., a mucosal membrane, gastrointestinal tract, injury site, organ, etc.) of a host.

[0233] 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).

[0234] Derivative: As used herein, the term “derivative”, or grammatical equivalents thereof, refers to a structural analogue of a reference substance. That is, a “derivative” is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. Such a substance would be said to be “derived from” said reference substance. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.

[0235] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, α-helix character, β-sheet character, coiled-coil character, random coil 58 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0236] 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.

[0237] 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).

[0238] 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 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 59 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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%, or 99% 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.

[0239] 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.

[0240] 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 some 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%, at least 99.5%, 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 60 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0241] 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.

[0242] Immunologically effective amount or immunologically effective dose: As used herein, “immunologically effective amount” or “immunologically effective dose” refers to an amount of an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, which when administered to a subject, either in a single dose or as part of a series of doses, that is sufficient to enhance a subject’s own immune response against a subsequent exposure to a pathogen. In some embodiments, the pathogen is S. agalactiae (Group B strep). In some embodiments, the immune response is against one or more different serotypes of S. agalactiae (Group B strep). In some embodiments, the immune response is against two or more different serotypes of S. agalactiae (Group B strep). In some embodiments, the immune response is against four or more different serotypes of S. agalactiae (Group B strep). In some embodiments, the immune response is against five or more different serotypes of S. agalactiae i some embodiments, the immune response is against six or more different serotypes of S. agalactiae (Group B strep). In some embodiments, the immune response is against seven or more different serotypes of S. agalactiae (Group B strep). In some embodiments, the immune response is against eight or more different serotypes of S. agalactiae (Group B strep). An immunologically effective amount may vary based on the subject to be treated, the species of the subject, the degree of immune response desired to induce, etc. In some embodiments, an immunologically effective amount is sufficient for treatment or protection of a subject having or at risk of having disease. In some embodiments, an immunologically effective amount refers to a non-toxic but sufficient amount that can be an amount to treat, attenuate, or prevent infection and / or disease (e.g., bacterial infection, S. agalactiae infection, bacterial colonization, S. agalactiae colonization, complications associated with bacterial infection, complications associated with S. agalactiae infection, etc.) in any subject. In some embodiments, an immunologically effective amount is sufficient to induce an immunoprotective response upon administration to a subject. 61 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0243] 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 naïve subject, efficient clearance of the activating agent or pathogen, followed by rapid resolution of inflammation. In some embodiments, an immunoprotective response is an adaptive immune response. In some embodiments, an immunoprotective response is sufficient to protect an immunized subject from productive infection by a particular pathogen or pathogens to which a vaccine is directed (e.g., S. agalactiae (Group B strep) infection).

[0244] 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.

[0245] 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%, 62 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0246] Linker: As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 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).

[0247] Pathogen: As used herein the term “pathogens” may include, for example, viral, bacterial, protozoan and / or fungal pathogens, some of which may possess an ability to interact and / or associate with and / or bind to, a host cell sialic acid containing receptor. Thus the vaccine composition as disclosed herein is useful on an application in the treatment and / or prevention of respiratory diseases / infections caused or contributed to by viral, bacterial, protozoan and / or fungal pathogens, some 63 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT of which may exploit the presence of sialic acid containing receptors on the surface of epithelial cells lining the surface of the upper and lower respiratory tracts.

[0248] 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 non- aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0249] 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.

[0250] Plurality: As used herein, the term “plurality” includes at least 2 or more, including, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more.

[0251] 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).

[0252] 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 64 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0253] 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 predefined period of time.

[0254] 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.

[0255] 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; 65 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.).

[0256] 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.

[0257] Response: As used herein, a “response” to treatment may refer to any beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment. Such alteration may include stabilization of the condition (e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and / or improvement in the prospects for cure of the condition, etc. It may refer to a subject’s response or to a tumor’s response. Subject or tumor response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of biomarkers in a sample obtained from a subject, cytology, and / or histology. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of subjects and / or tumors, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.

[0258] 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 66 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0259] 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.

[0260] 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. Thus, the “subject” (whether animal or human), may be symptomatic and / or (suspected of) suffering from an infection or disease by a pathogen as disclosed herein, for example a respiratory infection or disease. Alternatively, the subject may be asymptomatic and / or predisposed / susceptible to an infection or disease by the pathogen, for example, a respiratory infection or disease. In all cases, the infection or disease may have a microbial (for example bacterial, viral, protozoan and / or fungal aetiology.

[0261] 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).

[0262] 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, 67 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 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.

[0263] 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.

[0264] 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 some 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. The terms "vaccine" or "vaccine composition", which are used interchangeably, refer to pharmaceutical compositions comprising at least one immunogenic composition, e.g., SBD and polysaccharide, that induces an immune response in an animal. REFERENCES

[0265] All the references cited herein and throughout the application are incorporated by reference in their entirety. EQUIVALENTS AND SCOPE

[0266] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0267] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly 68 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0268] It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0269] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0270] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0271] Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses. 69 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT

[0272] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0273] The inventions provided herein are further described in the following numbered paragraphs: 1. The vaccine composition comprising at an antigenic polysaccharide and an immunomodulatory amount of a sialic acid binding moiety, wherein the sialic acid binding moiety comprises sialic acid binding domain (SBD) which is not fused to an antigenic polypeptide. 2. The vaccine composition of paragraph 1, wherein the SBD is selected from any of: SBD1, SBD2, SBD3, SBD4, NanH, NanH2, NanH3,VcNanH, or a SBD comprising an amino acid sequence that at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of: SEQ ID NO: 1-10. 3. The vaccine composition of any of the preceding paragraphs, wherein the SBD is selected from: i. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 1 (SBD1), or an amino acid having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 (SBD1), ii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 2 (SBD2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 2 (SBD2), iii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 3 (SBD3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 (SBD3), iv. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 4 (SBD4), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 4 (SBD4), v. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 5 (NanH), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 5 (NanH), 70 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT vi. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 6 (NanH2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 6 (NanH2), vii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 7 (NanH3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 7 (NanH3), or viii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 10 (NanH Vibrio Cholera), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 10 (NanH Vibrio Cholera) 4. The vaccine composition of any of the preceding paragraphs, wherein the SBD is lipidated. 5. The vaccine composition of any of the preceding paragraphs, wherein the lipidated SBD protein comprises at the N-terminus of the SBD protein, a lipidation sequence selected from any of: MKKVAAFVALSLLMAGC (SEQ ID NO: 16); MNSKKLCCICVLFSLLAGCAS (SEQ ID NO: 17), MRYSKLTMLIPCALLLSAC (SEQ ID NO: 18), MFVTSKKMTAAVLAITLAMSLSAC (SEQ ID NO: 19) and MIKRVLVVSMVGLSLVGC (SEQ ID NO: 20) or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of SEQ ID NO: 16-20. 6. The vaccine composition of any of the preceding paragraphs, wherein the antigenic polysaccharide comprises sialic acid on the surface. 7. The vaccine composition of any of the preceding paragraphs, wherein the antigenic polysaccharide does not comprises sialic acid on the surface. 8. The vaccine composition of any of the preceding paragraphs, wherein the immunomodulatory amount of a Sialic acid binding moiety is a ratio of an amount of SBD to the amount of antigenic polysaccharide to suppress the immune tolerance of the antigenic polysaccharide in a subject, as compared to the immune response to the antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety. 9. The vaccine composition of any of the preceding paragraphs, wherein the immunomodulatory amount of a Sialic acid binding moiety is a ratio of an amount of SBD to the amount of antigenic polysaccharide to enhance the immune response of the antigenic polysaccharide in a subject, as compared to the immune response to the 71 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety. 10. The vaccine composition of any of the preceding paragraphs, wherein the immunomodulatory amount of a Sialic acid binding moiety is the amount of SBD to bind to at least 20%, or 30%, or 40%, or 50%, or 60%, or 70% or more than 70% of sialic acids molecules present on the antigenic polysaccharide. 11. The vaccine composition of any of the preceding paragraphs, wherein the w / w ratio of SBD protein:antigenic polysaccharide present in a vaccine is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 or more than 20:1. 12. The vaccine composition of any of the preceding paragraphs, wherein the ratio of SBD to sialic acid molecules present on the antigenic polysaccharide is about 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3.0, 1:3.5, 1:4.0, 1:4.5, 1:5.0 or more than 5:1. 13. The vaccine composition of any of the preceding paragraphs, wherein the antigenic polysaccharide is selected from the group consisting of: polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-positive bacteria; polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-negative bacteria; other bacterial capsular or cell wall polysaccharides; fungal polysaccharides; viral polysaccharides; and polysaccharides derived from cancer or tumor cells. 14. The vaccine composition of any of the preceding paragraphs, wherein the antigenic polysaccharide is a capsular polysaccharide from a pathogen and has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%. 15. The vaccine composition of any of the preceding paragraphs, wherein the capsular polysaccharide has about 1.0 mM sialic acid per mM of polysaccharide, such as at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM of polysaccharide. 16. The vaccine composition of any of the preceding paragraphs, wherein the capsular polysaccharide is selected from any of: Salmonella typhi Vi capsular polysaccharides; Salmonella polysaccharides; Shigella polysaccharide, pneumococcal polysaccharides; Haemophili polysaccharides; Meningococcal polysaccharides; Staphylococcus aureus polysaccharides; Bacillus anthracis polysaccharides; Streptococcus polysaccharides; Pseudomonas polysaccharides; Cryptococcus polysaccharides; and viral glycoproteins. 17. The vaccine composition of any of the preceding paragraphs, wherein the antigenic polysaccharide exists as a polysaccharide-protein conjugate, including immunogenic polysaccharide-protein conjugates where the protein is covalently bound to the polysaccharide or non-covalently associated with the polysaccharide. 72 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 18. The vaccine composition of any of the preceding paragraphs, wherein the immunogenic polysaccharide-protein conjugate is a multiple antigen presenting system (MAPS) as disclosed in any of patent applications: WO / 2020 / 056127, WO / 2020 / 056202, WO / 2014 / 124228, WO / 2023 / 039223, US11560410B2, WO / 2018 / 183475, WO / 2018 / 217564, WO / 2023 / 102359, WO / 2023 / 102359A9, WO / 2012 / 155007, WO / 2023 / 192997A2WO / 2013 / 134656, WO / 2023 / 039108, WO / 2012 / 155053, WO / 2023 / 172741A2, WO / 2014 / 124228, WO / 2020 / 056127, WO / 2017 / 192801, WO / 2020 / 056202, WO / 2018 / 237221, WO / 2023 / 039223, US11305001, US20220362367, US20210008192, US20200121777, US20140154287, US10766932, US20210332090, US20230233667, US11560410, US10611805, US20160090404, US10017548, US9499593, US20140154286, US20200407404, US20190119335, US20150374811, US11576958, US20230081705, US20230089151, US20200087361, US20190119332, US11013793, US11701416, US20210346487, US20200222522, US11612647, US20220072118, US20230091255, or US20150374811, or WO2023 / 102359 or PCT applications: PCT / 2023 / 76822 and PCT / 2023 / 76878 19. A method to induce an immune response in a subject comprising administering any of the vaccine composition of any of the preceding paragraphs, wherein the immune response to the polysaccharide in the vaccine is greater as compared to the immune response to the same polysaccharide in the absence of the presence of a sialic acid binding protein (SBD). 73 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT SEQUENCES SEQ ID NO: 1: SBD1 VIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYN NTATLEGRGSDGKQFYNNYNDAPLKVKUGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFI KDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS SEQ ID NO: 2: >SBD2 NanL (81-272) IPEGILMEKNNVDIAEGQGYSLDQEAGAKYVKAMTQGTIILSYKSTSENGIQSLFSVGNSTAGNQDRHF HIYITNSGGIGIELRNTDGVFNYTLDRPASVRALYKGERVFNTVALKADAANKQCRLFANGELLATLDK DAFKFISDITGVDNVTLGGTKRQGKIAYPFGGTIGDIKVYSNALSDEELIQATG SEQ ID NO: 3: >SBD3 NanB (40-230) SPIFQGGSYQLNNKSIDISSLLLDKLSGESQTVVMKFKADKPNSLQALFGLSNSKAGFKNNYFSIFMRD SGEIGVEIRDAQKGINYLFSRPASLWGKHKGQAVENTLVFVSDSKDKTYTMYVNGIEVFSETVDTFLPI SNINGIDKATLGAVNREGKEHYLAKGSIDEISLFNKAISDQEVSTIPLSNPFQ SEQ ID NO: 4: >SBD4 NanC 82-270 EETPVLEKNNVTLTGGGENVTKELKDKFTSGDFTVVIKYNQSSEKGLQALFGISNSKUGQQNSYVDVFL RDNGELGMEARDTSSNKNNLVSRPASVWGKYKQEAVTNTVAVVADSVKKTYSLYANGTKVVEKKVDNFL NIKDIKGIDYYMLGGVKRAGKTAFGFNGTLENIKFFNSALDEETVKKMTTN SEQ ID NO: 5: >NanH Salmonella TVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAA ARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTD WDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTS FIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRN HGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDDFYPKVGNASGAGYSCLS YRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYN SEQ ID NO: 6:>NanH2 MKKFIKILKVLSMAIVLSACNINGIFASNLNTTNEPQKTTVFNKNDNTWNAQYFRIPSLQTLADGTMLA FSDIRYNGAEDHAYIDIGAAKSTDNGQTWDYKTVMENDRIDSTFSRVMDSTTVVTDTGRIILIAGSWNK NGNWASSTTSLRSDWSVQMVYSDDNGETWSDKVDLTTNKARIKNQPSNTIGWLAGVGSGIVMSDGTIVM PIQIALRENNANNYYSSVIYSKDNGETWTMGNKVPDPKTSENMVIELDGALIMSSRNDGKNYRASYISY DMGSTWEVYDPLHNKISTGNGSGCQGSFIKVTAKDGHRLGFISAPKNTKGGYVRDNITVYMIDFDDLSK GIRELCSPYPEDGNSSGGGYSCLSFNDGKLSILYEANGNIEYKDLTDYYLSIENNKKLK 74 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT SEQ ID NO: 7: >>NanH3 Trypanosoma cruzi 4-399 GSSRVELFKRQSSKVPFEKDGKVTERVVHSFRLPALVNVDGVMVAIADARYETSFDNSLIDTVAKYSVD DGETWETQIAIKNSRASSVSRVVDPTVIVKGNKLYVLVGSYNSSRSYWTSHGDARDWDILLAVGEVTKS TAGGKITASIKWGSPVSLKEFFPAEMEGMHTNQFLGGAGVAIVASNGNLVYPVQVTNKKKQVFSKIFYS EDEGKTWKFGKGRSAFGCSEPVALEWEGKLIINTRVDYRRRLVYESSDMGNTWLEAVGTLSRVWGPSPK SNQUGSQSSFTAVTIEGMRVMLFTHPLNFKGRWLRDRLNLWLTDNQRIYNVGQVSIGDENSAYSSVLYK DDKLYCLHEINSNEVYSLVFARLVGELRIIKSVLQSWKNWDSHLSSICTPA SEQ ID NO: 8: Streptococcus pneumoniae NanA sialidase amino acid sequence MSYFRNRDIDIERNSMNRSVQERKCRYSIRKLSVGAVSMIVGAVVFGTSPVLAQEGASEQPLANETQLS GESSTLTDTEKSQPSSETELSGNKQEQERKDKQEEKIPRDYYARDLENVETVIEKEDVETNASNGQRVD LSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNN YNDAPLKVKUGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRAN NTVWGSNLQIRNLTVYNRALTPEEVQKRSQLFKRSDLEKKLPEGAALTEKTDIFESGRNGKPNKDGIKS YRIPALLKTDKGTLIAGADERRLHSSDWGDIGMVIRRSEDNGKTWGDRVTITNLRDNPKASDPSIGSPV NIDMVLVQDPETKRIFSIYDMFPEGKGIFGMSSQKEEAYKKIDGKTYQILYREGEKGAYTIRENGTVYT PDGKATDYRVVVDPVKPAYSDKGDLYKGNQLLGNIYFTTNKTSPFRIAKDSYLWMSYSDDDGKTWSAPQ DITPMVKADWMKFLGVGUGTGIVLRNGPHKGRILIPVYTTNNVSHLNGSQSSRIIYSDDHGKTWHAGEA VNDNRQVDGQKIHSSTMNNRRAQNTESTVVQLNNGDVKLFMRGLTGDLQVATSKDGGVTWEKDIKRYPQ VKDVYVQMSAIHTMHEGKEYIILSNAGGPKRENGMVHLARVEENGELTWLKHNPIQKGEFAYNSLQELG NGEYGILYEHTEKGQNAYTLSFRKFNWDFLSKDLISPTEAKVKRTREMGKGVIGLEFDSEVLVNKAPTL QLANGKTARFMTQYDTKTLLFTVDSEDMGQKVTGLAEGAIESMHNLPVSVAGTKLSNGMNGSEAAVHEV PEYTGPLGTSGEEPAPTVEKPEYTGPLGTSGEEPAPTVEKPEYTGPLGTAGEEAAPTVEKPEFTGGVNG TEPAVHEIAEYKGSDSLVTLTTKEDYTYKAPLAQQALPETGNKESDLLASLGLTAFFLGLFTLGKKREQ SEQ ID NO: 9: Vibrio cholerae NanH sialidase amino acid sequence MRFKNVKKTALMLAMFGMATSSNAALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQ GIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLVVEFEGQ TGRTVLATGTAATEYHKFELVFLUGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYR DIKFEIQGDVIFRGPDRIPSIVASSVTUGVVTAFAEKRVGGGDUGALSNTNDIITRTSRDGGITWDTEL NLTEQINVSDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRIKPWMPNGIFYSVYDVASGNWQAP IDVTDQVKERSFQIAGWGGSELYRRNTSLNSQQDWQSNAKIRIVDGAANQIQVADGSRKYVVTLSIDES GGLVANLNGVSAPIILQSEHAKVHSFHDYELQYSALNHTTTLFVDGQQITTWAGEVSQENNIQFGNADA QIDGRLHVQKIVLTQQGHNLVEFDAFYLAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASVNUGUGHGI TLTRQQNISGSQNGRLIYPAIVLDRFFLNVMSIYSDDGGSNWQTGSTLPIPFRWKSSSILETLEPSEAD MVELQNGDLLLTARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNISTGTVDASITRFEQ 75 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT SDGSHFLLFTNPQGNPAGTNGRQNLGLWFSFDEGVTWKGPIQLVNGASAYSDIYQLDSENAIVIVETDN SNMRILRMPITLLKQKLTLSQN SEQ ID NO: 10: SBD of Vibrio cholerae NanH sialidase (aa 25-216 of SEQ ID NO: 113) ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFG WRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLU GSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD SEQ ID NO:11 nucleic acid encoding SBD1 GTGATAGAAAAAGAAGATGTTGAAACCAATGCTTCAAATGGTCAGAGAGTTGATTTATCAAGTGAACTA GATAAACTAAAGAAACTTGAAAACGCAACAGTTCACATGGAGTTTAAGCCAGATGCCAAGGCCCCAGCA TTCTATAATCTCTTTTCTGTGTCAAGTGCTACTAAAAAAGATGAGTACTTCACTATGGCAGTTTACAAT AATACTGCTACTCTAGAGGGGCGTGGTTCGGATGGGAAACAGTTTTACAATAATTACAACGATGCACCC TTAAAAGTTAAACCAGGTCAGTGGAATTCTGTGACTTTCACAGTTGAAAAACCGACAGCAGAACTACCT AAAGGCCGAGTGCGCCTCTACGTAAACGGGGTATTATCTCGAACAAGTCTGAGATCTGGCAATTTCATT AAAGATATGCCAGATGTAACGCATGTGCAAATCGGAGCAACCAAGCGTGCCAACAATACGGTTTGGGGG TCAAATCTACAGATTCGGAATCTCACTGTGTATAATCGTGCTTTAACACCAGAAGAGGTACAAAAACGT AGT SEQ ID NO:12 nucleic acid encoding SBD2 ATTCCGGAAGGCATTCTGATGGAAAAGAACAATGTTGATATAGCCGAAGGGCAGGGATACAGCCTGGAC CAGGAGGCTGGCGCCAAATATGTAAAGGCTATGACCCAGGGTACAATTATCCTATCCTATAAATCAACA AGTGAAAATGGAATCCAGTCTCTGTTCAGCGTAGGAAACAGTACCGCGGGCAATCAGGACAGGCATTTC CACATCTATATAACAAACTCTGGCGGCATAGGCATAGAACTTAGAAACACGGACGGTGTCTTCAATTAT ACATTGGACAGACCGGCATCAGTGCGTGCTCTGTATAAAGGAGAACGGGTATTCAATACAGTTGCATTA AAAGCCGATGCAGCAAATAAACAGTGCAGATTATTTGCAAATGGCGAGCTTCTCGCAACACTGGATAAA GATGCGTTTAAGTTTATCAGTGACATTACAGGCGTAGATAACGTTACATTAGGCGGCACGAAGAGGCAG GGGAAGATTGCATATCCGTTCGGGGGAACGATTGGTGACATTAAGGTATATAGTAATGCACTGTCCGAC GAAGAATTGATACAGGCAACAGGA SEQ ID NO:13 nucleic acid encoding SBD2 (codon-optimized) ATTCCCGAAGGCATCCTGATGGAGAAAAATAACGTTGATATTGCCGAGGGCCAGGGCTATTCTTTGGAT CAAGAGGCCGGTGCCAAGTATGTAAAAGCAATGACACAAGGGACCATTATTCTGTCATACAAATCGACC TCAGAGAATGGCATCCAATCGTTATTCTCAGTCGGGAATAGTACTGCTGGTAATCAGGACCGTCATTTT CATATCTATATTACAAACTCGGGGGGCATTGGAATCGAGCTTCGTAACACAGATGGCGTATTCAACTAT 76 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT ACTTTGGACCGCCCGGCGTCTGTTCGCGCGCTGTATAAAGGGGAGCGCGTGTTCAATACGGTTGCTTTG AAGGCGGACGCCGCCAATAAGCAGTGTCGCTTGTTCGCAAATGGCGAATTGTTAGCTACGCTTGACAAG GACGCATTTAAGTTTATTTCCGACATCACTGGGGTGGACAATGTAACGTTGGGTGGAACCAAACGCCAG GGCAAAATTGCATACCCGTTCGGAGGGACAATCGGCGACATTAAAGTCTATAGCAACGCGCTGAGTGAC GAAGAGCTTATCCAGGCTACCGGA SEQ ID NO:14 nucleic acid encoding SBD3 TCTCCTATTTTTCAAGGAGGTTCATATCAACTGAACAATAAGAGTATAGATATCAGCTCTTTGTTATTA GATAAATTGTCTGGAGAGAGTCAGACAGTAGTAATGAAATTTAAAGCAGATAAACCAAACTCTCTTCAA GCTTTGTTTGGCCTATCTAATAGTAAAGCAGGCTTTAAAAATAATTACTTTTCAATTTTCATGAGAGAT TCTGGTGAGATAGGTGTAGAAATAAGAGACGCCCAAAAGGGAATAAATTATTTATTTTCCAGACCAGCT TCATTATGGGGAAAACATAAAGGACAGGCAGTTGAAAATACACTAGTATTTGTATCTGATTCTAAAGAT AAAACATACACAATGTATGTTAATGGAATAGAAGTGTTCTCTGAAACAGTTGATACATTTTTGCCAATT TCAAATATAAATGGTATAGATAAGGCAACACTAGGAGCTGTTAATCGTGAAGGTAAGGAACATTACCTC GCAAAAGGAAGTATTGATGAAATCAGTCTATTTAACAAAGCAATTAGTGATCAGGAAGTTTCAACTATT CCCTTGTCAAATCCATTTCAG SEQ ID NO:15 nucleic acid encoding SBD4 GAAGAAACTCCTGTCTTAGAAAAAAATAATGTTACTTTAACAGGGGGCGGAGAAAATGTTACTAAAGAG TTAAAGGATAAATTTACTAGCGGTGACTTTACTGTAGTGATTAAGTACAATCAGTCAAGTGAGAAAGGC TTACAAGCTCTGTTTGGAATATCTAATTCCAAACCCGGTCAACAAAATAGTTATGTAGATGTGTTCCTT AGAGACAATGGTGAGTTGGGGATGGAAGCGCGTGATACTTCTTCCAATAAAAATAACCTAGTATCCAGA CCTGCTTCAGTTTGGGGTAAGTACAAACAAGAGGCTGTGACTAACACTGTTGCAGTAGTAGCAGATTCA GTCAAAAAAACATATTCTTTATACGCAAATGGTACAAAAGTAGTAGAAAAGAAAGTGGATAATTTCCTA AACATCAAGGATATTAAAGGTATTGATTACTATATGCTTGGGGGAGTGAAACGTGCAGGAAAAACGGCG TTTGGTTTTAACGGAACACTAGAAAATATCAAATTCTTTAATAGTGCATTGGATGAAGAAACTGTTAAA AAGATGACAACAAAC 77 4854-5196-4398.1 701039-000134USPL

Claims

Attorney Docket: 701039-000134WOPT CLAIMS 1. The vaccine composition comprising at an antigenic polysaccharide and an immunomodulatory amount of a sialic acid binding moiety, wherein the sialic acid binding moiety comprises sialic acid binding domain (SBD) which is not fused to an antigenic polypeptide.

2. The vaccine composition of claim 1, wherein the SBD is selected from any of: SBD1, SBD2, SBD3, SBD4, NanH, NanH2, NanH3,VcNanH, or a SBD comprising an amino acid sequence that at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of: SEQ ID NO: 1-10.

3. The vaccine composition of any of claims 1-2, wherein the SBD is selected from: i. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 1 (SBD1), or an amino acid having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 (SBD1), ii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 2 (SBD2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 2 (SBD2), iii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 3 (SBD3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 3 (SBD3), iv. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 4 (SBD4), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 4 (SBD4), v. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 5 (NanH), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 5 (NanH), vi. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 6 (NanH2), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 6 (NanH2), vii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 7 (NanH3), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 7 (NanH3), or viii. a polypeptide comprising, or consisting of an amino acid of SEQ ID NO: 10 (NanH Vibrio Cholera), or an amino acid having an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to SEQ ID NO: 10 (NanH Vibrio Cholera) 4. The vaccine composition of any of claims 1-3, wherein the SBD is lipidated. 78 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 5. The vaccine composition of claim 4, wherein the lipidated SBD protein comprises at the N- terminus of the SBD protein, a lipidation sequence selected from any of: MKKVAAFVALSLLMAGC (SEQ ID NO: 16); MNSKKLCCICVLFSLLAGCAS (SEQ ID NO: 17), MRYSKLTMLIPCALLLSAC (SEQ ID NO: 18), MFVTSKKMTAAVLAITLAMSLSAC (SEQ ID NO: 19) and MIKRVLVVSMVGLSLVGC (SEQ ID NO: 20) or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to any of SEQ ID NO: 16-20.

6. The vaccine composition of any of claims 1-5, wherein the antigenic polysaccharide comprises sialic acid on the surface.

7. The vaccine composition of any of claims 1-6, wherein the antigenic polysaccharide does not comprises sialic acid on the surface.

8. The vaccine composition of any of claims 1-7, wherein the immunomodulatory amount of a Sialic acid binding moiety is a ratio of an amount of SBD to the amount of antigenic polysaccharide to suppress the immune tolerance of the antigenic polysaccharide in a subject, as compared to the immune response to the antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety.

9. The vaccine composition of any of claims 1-8, wherein the immunomodulatory amount of a Sialic acid binding moiety is a ratio of an amount of SBD to the amount of antigenic polysaccharide to enhance the immune response of the antigenic polysaccharide in a subject, as compared to the immune response to the antigenic polysaccharide in the absence of an immunomodulatory amount of a Sialic acid binding moiety.

10. The vaccine composition of any of claims 1-9, wherein the immunomodulatory amount of a Sialic acid binding moiety is the amount of SBD to bind to at least 20%, or 30%, or 40%, or 50%, or 60%, or 70% or more than 70% of sialic acids molecules present on the antigenic polysaccharide.

11. The vaccine composition of any of claims 1-9, wherein the w / w ratio of SBD protein:antigenic polysaccharide present in a vaccine is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 or more than 20:

1.

12. The vaccine composition of any of claims 1-9, wherein the ratio of SBD to sialic acid molecules present on the antigenic polysaccharide is about 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3.0, 1:3.5, 1:4.0, 1:4.5, 1:5.0 or more than 5:

1.

13. The vaccine composition of any of claims 1-12, wherein the antigenic polysaccharide is selected from the group consisting of: polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-positive bacteria; polysaccharides, oligosaccharides, or lipopolysaccharides from Gram-negative bacteria; other bacterial capsular or cell wall polysaccharides; fungal polysaccharides; viral polysaccharides; and polysaccharides derived from cancer or tumor cells. 79 4854-5196-4398.1 701039-000134USPLAttorney Docket: 701039-000134WOPT 14. The vaccine composition of any of claims 1-12, wherein the antigenic polysaccharide is a capsular polysaccharide from a pathogen and has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

15. The vaccine composition of any of claims 1-14, wherein the capsular polysaccharide has about 1.0 mM sialic acid per mM of polysaccharide, such as at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM of polysaccharide.

16. The vaccine composition of any of claims 1-15, wherein the capsular polysaccharide is selected from any of: Salmonella typhi Vi capsular polysaccharides; Salmonella polysaccharides; Shigella polysaccharide, pneumococcal polysaccharides; Haemophili polysaccharides; Meningococcal polysaccharides; Staphylococcus aureus polysaccharides; Bacillus anthracis polysaccharides; Streptococcus polysaccharides; Pseudomonas polysaccharides; Cryptococcus polysaccharides; and viral glycoproteins.

17. The vaccine composition of any of claims 1-16, wherein the antigenic polysaccharide exists as a polysaccharide-protein conjugate, including immunogenic polysaccharide-protein conjugates where the protein is covalently bound to the polysaccharide or non-covalently associated with the polysaccharide.

18. The vaccine composition of any of claims 1-16, wherein the immunogenic polysaccharide- protein conjugate is a multiple antigen presenting system (MAPS) as disclosed in any of patent applications: WO / 2020 / 056127, WO / 2020 / 056202, WO / 2014 / 124228, WO / 2023 / 039223, US11560410B2, WO / 2018 / 183475, WO / 2018 / 217564, WO / 2023 / 102359, WO / 2023 / 102359A9, WO / 2012 / 155007, WO / 2023 / 192997A2WO / 2013 / 134656, WO / 2023 / 039108, WO / 2012 / 155053, WO / 2023 / 172741A2, WO / 2014 / 124228, WO / 2020 / 056127, WO / 2017 / 192801, WO / 2020 / 056202, WO / 2018 / 237221, WO / 2023 / 039223, US11305001, US20220362367, US20210008192, US20200121777, US20140154287, US10766932, US20210332090, US20230233667, US11560410, US10611805, US20160090404, US10017548, US9499593, US20140154286, US20200407404, US20190119335, US20150374811, US11576958, US20230081705, US20230089151, US20200087361, US20190119332, US11013793, US11701416, US20210346487, US20200222522, US11612647, US20220072118, US20230091255, or US20150374811, or WO2023 / 102359 or PCT applications: PCT / 2023 / 76822 and PCT / 2023 / 76878 19. A method to induce an immune response in a subject comprising administering the vaccine composition of any of claims 1-18, wherein the immune response to the polysaccharide in the vaccine is greater as compared to the immune response to the same polysaccharide in the absence of the presence of a sialic acid binding protein (SBD). 80 4854-5196-4398.1 701039-000134USPL

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