Immunogenic compositions

An immunogenic composition comprising:

• (i) a backbone polymer comprising a polymer and one or more antigenic polysaccharides conjugated to the polymer,
• wherein the polymer is characterized by an average molecular weight of about 50 kDa to about 2000 kDa, and
• wherein the one or more antigenic polysaccharides are or comprise O-antigen polysaccharides (OPS) characterized by an average molecular weight of about 10 kDa to about 30 kDa;
• and
• (ii) one or more polypeptide antigens non-covalently complexed with the polymer and/or the antigenic polysaccharide(s).

A method of purifying an O-antigen polysaccharide, the method comprising steps of:

• (A) contacting Gram-negative bacteria or one or more cellular components thereof, optionally wherein the Gram-negative bacteria is or comprises Klebsiella, with an oxidizing reagent and an acid to obtain a mixture, wherein the pH of the mixture is between about 3 and 5, and wherein the oxidizing agent is sodium nitrite and the acid is acetic acid;
• separating the O-antigen polysaccharide from the cellular components of the Gram-negative bacteria;
• and
• recovering the OPS, wherein the OPS is substantially free of the cellular components and contains a 2,5-anhydromannose residue containing a free aldehyde at its reducing terminal end;
• or
• (B) contacting Gram-negative bacteria with an acidic reagent to obtain a mixture, wherein the pH of the mixture is between about 3 and 5,
• heating the mixture to between about 80° C. to about 100° C.;
• separating the O-antigen polysaccharide from cellular components of the Gram-negative bacteria;
• and
• recovering the OPS, wherein the OPS is substantially free of the cellular components and contains a ketone at its reducing terminal end.

A method of making a backbone polymer, wherein the backbone polymer comprises:

• (A) at least one non-biotinylated O-antigen polysaccharide, wherein the O-antigen polysaccharide comprises a primary amino group, and at least one biotinylated polysaccharide, the method comprising steps of:
• chemically linking the O-antigen polysaccharide primary amino group by mixing the O-antigen polysaccharide with a partially oxidized polysaccharide containing aldehyde groups and a biotin containing primary amine to obtain a mixture;
• and reductively aminating the mixture with a reducing agent to produce a BP-1 polymer backbone, wherein the polysaccharide is biotinylated and the chemically linked O-antigen polysaccharide is not biotinylated;
• or
• (B) at least one non-biotinylated O-antigen polysaccharide, wherein the O-antigen polysaccharide comprises a primary amino group, and at least one biotinylated polysaccharide, the method comprising steps of:
• chemically linking the O-antigen polysaccharide primary amino group by mixing the O-antigen polysaccharide with a polysaccharide biotinylated and oxidized obtained by first treating the carboxylate groups of the polysaccharide with 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide N-hydroxysuccinimide (EDC) and N-Hydroxysuccinimide (NHS) and a biotin hydrazide, then treating the polysaccharide with an oxidizing agent;
• and reductively aminating the mixture with a reducing agent to produce BP-1.2 polymer backbone, wherein the polysaccharide is biotinylated and the chemically linked O-antigen polysaccharide is not biotinylated;
• or
• (C) at least one non-biotinylated O-antigen polysaccharide, wherein the O-antigen polysaccharide comprises a primary amino group, and at least one biotinylated polysaccharide, the method comprising steps of:
• chemically linking the O-antigen polysaccharide primary amino group by mixing the O-antigen polysaccharide with a polysaccharide biotinylated and CDAP activated obtained by first treating the carboxylate groups of the polysaccharide with 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide N-hydroxysuccinimide (EDC) and N-Hydroxysuccinimide (NHS) and a biotin hydrazide, then treating the polysaccharide with CDAP to produce BP-1.3 polymer backbone, wherein the polysaccharide is biotinylated and the chemically linked O-antigen polysaccharide is not biotinylated;
• or
• (D) at least one non-biotinylated O-antigen polysaccharide, wherein the O-antigen polysaccharide comprises an azido group at its reducing terminal end, and at least one biotinylated polysaccharide containing alkynes groups, the method comprising a step of:
• chemically click-linking in the presence of a catalyst the O-antigen polysaccharide containing azido groups at their reducing ends with a biotinylated polysaccharide containing alkynes, thereby forming an OPS backbone polymer, wherein the polysaccharide is biotinylated but the chemically click-linked O-antigen polysaccharide is not biotinylated.

A fusion protein comprising:

• a biotin-binding domain and one of the following:
• (A) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of any one of SEQ ID NOs:1-3, 6-12, or 16-26;
• or
• (B) (i) a Pseudomonas flagellin B D2 domain (FlaBD2) polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:10 or an immunogenic fragment thereof, and (ii) a Klebsiella MrkA polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3 or an immunogenic fragment thereof;
• or
• (C) (i) a Pseudomonas flagellin B D2 domain (FlaBD2) polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:10 or an immunogenic fragment thereof, and (ii) a Pseudomonas PcrV polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:8 or an immunogenic fragment thereof.