Immunogenic compositions

Abstract

Technologies for the prevention and/or treatment of nosocomial infections.

1 - 135 . (canceled) 
     
     
         136 . 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).   
     
     
         137 . The immunogenic composition of  claim 136 , wherein the polymer is characterized by an average molecular weight of about 220 kDa. 
     
     
         138 . The immunogenic composition of  claim 136 , wherein the polymer is or comprises:
 a capsular polysaccharide derived from a Gram-negative or Gram-positive bacterium, or   (ii) a linear poly-L-lysine or a dendrimer of L-lysine.   
     
     
         139 . The immunogenic composition of  claim 138 , wherein the backbone polymer comprises at least one of the following features:
 (A) the polymer is or comprises:
 (i) a  Klebsiella  capsular polysaccharide,  Pseudomonas  exopolysaccharide, and/or  Escherichia  capsular polysaccharide; and/or 
 (ii) a  Klebsiella  spp. K19 capsular polysaccharide; and/or 
   (B) the OPS is or comprises a polysaccharide of one or more of  Klebsiella, Pseudomonas , and  E. coli.      
     
     
         140 . The immunogenic composition of  claim 139 , wherein the OPS is or comprises one or more of the following:
 (i) a  K. pneumoniae  OPS of type O1, O2, O3, O5, or combinations thereof; and   (ii) a  P. aeruginosa  OPS of type O1, O2, O3, O4, O5, O6, O10, O11, O12, or combinations thereof.   
     
     
         141 . The immunogenic composition of  claim 136 , wherein the one or more polypeptide antigens are non-covalently complexed with the polymer and/or the one or more antigenic polysaccharides through at least one affinity-molecule pair comprising (i) a first affinity molecule, and (ii) a second affinity molecule complementary to the first affinity molecule, wherein the first affinity molecule is associated with the polymer and/or the one or more antigenic polysaccharides; and wherein the second affinity molecule is associated with the one or more polypeptide antigens;
 optionally wherein the affinity-molecule pair is selected from the group consisting of: biotin/biotin-binding protein, antibody/antigen, enzyme/substrate, receptor/ligand, metal/metal-binding protein, carbohydrate/carbohydrate binding protein, lipid/lipid-binding protein, and His tag/His tag-binding molecule; and/or   optionally wherein the first affinity molecule is cross-linked or covalently bonded to the polymer and/or the one or more antigenic polysaccharides.   
     
     
         142 . The immunogenic composition of  claim 141 , wherein:
 (i) the first affinity molecule is or comprises biotin or a derivative thereof, and   (ii) the second affinity molecule is or comprises a biotin-binding protein or biotin-binding domain thereof, optionally wherein the second affinity molecule is or comprises rhizavidin or a biotin-binding domain thereof.   
     
     
         143 . The immunogenic composition of  claim 141 , wherein:
 (i) the polymer comprises a  Klebsiella  spp. K19 capsular polysaccharide;   (ii) the OPS comprises a polysaccharide of  Klebsiella  and/or a polysaccharide of  Pseudomonas ; and   (iii) the first affinity molecule is associated with the polymer.   
     
     
         144 . The immunogenic composition of  claim 141 , wherein the one or more polypeptide antigens are associated with the second affinity molecule in a fusion protein, optionally wherein the second affinity molecule is or comprises a biotin-binding protein or biotin-binding domain thereof. 
     
     
         145 . The immunogenic composition of  claim 144 , wherein the one or more polypeptide antigens comprise: a  Pseudomonas  flagellin polypeptide, a  Pseudomonas  PcrV polypeptide, a  Klebsiella  MrkA polypeptide, or combinations thereof. 
     
     
         146 . The immunogenic composition of  claim 144 , wherein the one or more polypeptide antigens comprise at least one of the following:
 (i) a  Pseudomonas  flagellin subtype B D2 domain (FlaBD2) polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:10, or an immunogenic fragment thereof;   (ii) a  Pseudomonas  flagellin subtype B (FlaB) polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7, or an immunogenic fragment thereof;   (iii) a  Pseudomonas  flagellin subtype A1 (FlaA1) D2 domain polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:11, or an immunogenic fragment thereof;   (iv) a  Pseudomonas  flagellin subtype A2 (FlaA2) D2 domain polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:12, or an immunogenic fragment thereof;   (v) a  Pseudomonas  PcrV polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:8, or an immunogenic fragment thereof; and   (vi) 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:2 or SEQ ID NO:3, or an immunogenic fragment thereof.   
     
     
         147 . The immunogenic composition of  claim 144 , wherein the one or more polypeptide antigens comprises at least two polypeptide antigens. 
     
     
         148 . The immunogenic composition of  claim 147 , wherein the at least two polypeptide antigens comprise:
 (A) (i) a  Pseudomonas  flagellin subtype B (FlaB) polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7, or an immunogenic fragment thereof, and (ii) a  Klebsiella  MrkA polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2 or SEQ ID NO:3, or an immunogenic fragment thereof;   (B) (i) a  Pseudomonas  flagellin subtype B D2 domain (FlaBD2) polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 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 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2 or SEQ ID NO:3, or an immunogenic fragment thereof;   (C) (i) a  Pseudomonas  flagellin subtype B (FlaB) polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7, or an immunogenic fragment thereof, and (ii) a  Pseudomonas  PcrV polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:8, or an immunogenic fragment thereof; or   (D) (i) a  Pseudomonas  flagellin subtype B D2 domain (FlaBD2) polypeptide lacking the TLR5 binding motif comprising an amino acid sequence having at least 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 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:8, or an immunogenic fragment thereof.   
     
     
         149 . The immunogenic composition of  claim 144 , wherein the fusion protein is or comprises:
 (i) a Rhavi-FlaBD2-MrkA fusion protein comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:24; or   (ii) a Rhavi-FlaBD2-PcrV fusion protein comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:26.   
     
     
         150 . The immunogenic composition of  claim 136 , wherein at least one of the polypeptide antigen(s) is or comprises:
 (i) a  Pseudomonas  polypeptide; or   (ii) a  Pseudomonas  flagellin polypeptide, a  Pseudomonas  PcrV polypeptide, a  Klebsiella  MrkA polypeptide, or combinations thereof; or   (iii) a  Pseudomonas  flagellin subtype A (FlaA) polypeptide, and/or a  Pseudomonas  flagellin subtype B (FlaB) polypeptide.   
     
     
         151 . The immunogenic composition of  claim 150 , wherein at least one of the polypeptide antigen(s) is or comprises:
 (i) 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:2 or SEQ ID NO:3, or an immunogenic fragment thereof;   (ii) a  Pseudomonas  flagellin subtype B (FlaB) 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:7, or an immunogenic fragment thereof;   (iii) a  Pseudomonas  flagellin subtype 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; or   (iv) 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.   
     
     
         152 . The immunogenic composition of  claim 136 , wherein the immunogenic composition is characterized in that upon administration to a subject, the immunogenic composition elicits an immune response against Gram-negative and/or Gram-positive bacteria;
 optionally wherein the Gram-negative bacteria are selected from  Klebsiella, Pseudomonas, E. coli , and a combination thereof; and/or   optionally wherein the immune response is or comprises:
 (i) an antibody or B cell response; and/or 
 (ii) a T cell response, optionally wherein the T cell response is or comprises a CD4+ T cell response (e.g., including Th1, Th2, or Th17 response) and/or a CD8+ T cell response. 
   
     
     
         153 . A vaccine comprising one or more species of the immunogenic composition of  claim 136 . 
     
     
         154 . The vaccine of  claim 153 , comprising a plurality of distinct species of the immunogenic composition of  claim 136 , optionally wherein the O-antigen polysaccharides of the plurality of species of the immunogenic composition comprise each of:
 (i) a  Klebsiella pneumoniae  OPS of type O1, O2, O3, and O5;   (ii) a  Pseudomonas aeruginosa  OPS of type O1, O2, O3, O4, O5, O6, O10, and 011;   or   (iii) a combination of (i) and (ii).   
     
     
         155 . A pharmaceutical composition comprising one or more species of the immunogenic composition of  claim 136  and a pharmaceutically acceptable carrier,
 optionally wherein the pharmaceutical composition is characterized by one or more of the following features:
 (i) the pharmaceutical composition is formulated for injection; 
 (ii) the pharmaceutical composition further comprises one or more adjuvants; optionally wherein the one or more adjuvants are selected from the group consisting of: aluminum phosphate, aluminum hydroxide, phosphated aluminum hydroxide, TLRs agonists (such as the TLR2 saponin (QS21) or porins, and TLR4 agonists such as for example monophosphoryl lipidA (MPL), and TLR5 such as flagellins, etc.); and 
 (iii) upon administration to a subject, the pharmaceutical composition:
 (a) elicits a Th1 and/or Th17 cell response; 
 (b) elicits an opsonic/bactericidal response against one or more pathogens, optionally wherein the one or more pathogens comprise  Klebsiella, Pseudomonas, E. coli , or combinations thereof; 
 (c) reduces rate of transmission and/or colonization of the mucosal surfaces by one or more pathogens, optionally wherein the one or more pathogens comprise  Klebsiella, Pseudomonas, E. coli , or combinations thereof; and/or 
 (d) reduces rate of transmission and/or colonization of the GI tract by one or more pathogens, optionally wherein the one or more pathogens comprise  Klebsiella, Pseudomonas, E. coli , or combinations thereof. 
 
 
 
     
     
         156 . A method of immunizing a subject against a nosocomial infection comprising administering to the subject an effective amount of the immunogenic composition of  claim 136 ,
 optionally wherein the nosocomial infection is or comprises  Klebsiella, Pseudomonas , and/or  E. coli  infection.   
     
     
         157 . 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. 
   
     
     
         158 . 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. 
   
     
     
         159 . 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.