Synthetic Oligosaccharide Subunits Of The PSL Exopolysaccharide Of Pseudomonas Aeruginosa And Uses Thereof

Abstract

This disclosure relates to synthetic oligosaccharide subunits of the Pseudomonas exosaccharide Psi and uses thereof, e.g., for epitope mapping of anti-Psl antibodies, for identification of anti-Psl antibodies, and for use as vaccines. In one aspect a synthetic oligosaccharide subunit of a Pseudomonas aeruginosa Psl oligosaccharide is provided, comprising the trisaccharide of formula I.

What is claimed is: 
     
         1 . A synthetic oligosaccharide subunit of a  Pseudomonas aeruginosa  Psl oligosaccharide, comprising the trisaccharide of formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  is —OH or 
       
       
         
           
           
               
               
           
         
       
       wherein R 2  is —OH or 
       
         
           
           
               
               
           
         
         wherein R 3  is —OX, where X is hydrogen, an alkyl group, or an aryl group; a linker, wherein the linker can be —O—(CH 2 ) n —NH 2 , —O—(CH 2 ) n —COOH, —O—(CH 2 ) n —N 3 , —O—(CH 2 ) n —S(CH 2 ) m —NH 2 , —O—(CH 2 ) n —S(CH 2 ) m —COOH, —O—(CH 2 ) n —S(CH 2 ) m —N 3 , —O—(CH 2 ) n —SO 2 (CH 2 ) m —NH 2 , —O—(CH 2 ) n —SO 2 (CH 2 ) m —COOH, or —O—(CH 2 ) n —SO 2 (CH 2 ) m —N 3 , wherein n and m are the same or different and are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or a linker coupled to a heterologous moiety via conjugation, wherein the heterologous moiety can be a protein, a lipid or a polymer; and 
         wherein the oligosaccharide subunit can be specifically bound by the anti-Psl monoclonal antibody WapR-016, or an antigen binding fragment thereof. 
       
     
     
         2 . The oligosaccharide subunit of  claim 1 , comprising the hexasaccharide: 
       
         
           
           
               
               
           
         
         or the decasaccharide: 
       
       
         
           
           
               
               
           
         
       
     
     
         3 . The oligosaccharide subunit of  claim 1  or  claim 2 , wherein monoclonal antibody WapR-016 comprises VH and VL amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12, respectively. 
     
     
         4 . A synthetic oligosaccharide subunit of a  Pseudomonas aeruginosa  Psl oligosaccharide, comprising the tetrasaccharide of formula II: 
       
         
           
           
               
               
           
         
         wherein R 4  is —OH or 
       
       
         
           
           
               
               
           
         
         wherein R 5  is —OH, 
       
       
         
           
           
               
               
           
         
         wherein R 3  is —OX, where X is hydrogen, an alkyl group, or an aryl group; a linker, wherein the linker can be —O—(CH 2 ) n —NH 2 , —O—(CH 2 ) n —COOH, —O—(CH 2 ) n —N 3 , —O—(CH 2 ) n —S(CH 2 ) m —NH 2 , —O—(CH 2 ) n —S(CH 2 ) m —COOH, —O—(CH 2 ) n —S(CH 2 ) m —N 3 , —O—(CH 2 ) n —SO 2 (CH 2 ) m —NH 2 , —O—(CH 2 ) n —SO 2 (CH 2 ) m —COOH, or —O—(CH 2 ) n —SO 2 (CH 2 ) m —N 3 , where n and m are the same or different and are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or a linker coupled to a heterologous moiety via conjugation, wherein the heterologous moiety can be a protein, a lipid or a polymer; and 
         wherein the oligosaccharide subunit can be specifically bound by the anti-Psl monoclonal antibody WapR-001, or an antigen binding fragment thereof. 
       
     
     
         5 . The oligosaccharide subunit of  claim 4 , comprising the tetrasaccharide of: 
       
         
           
           
               
               
           
         
         the pentasaccharide of: 
       
       
         
           
           
               
               
           
         
         the hexasaccharide of: 
       
       
         
           
           
               
               
           
         
         or the decasaccharide of: 
       
       
         
           
           
               
               
           
         
       
     
     
         6 . The oligosaccharide subunit of  claim 4  or  claim 5 , wherein monoclonal antibody WapR-001 comprises VH and VL amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, respectively. 
     
     
         7 . The oligosaccharide subunit of  claim 1 , wherein anti-Psl monoclonal antibody WapR-016, or an antigen binding fragment thereof cannot specifically bind to the tetrasaccharide of  claim 5  or the pentasaccharide of  claim 5 . 
     
     
         8 . The oligosaccharide subunit of any one of  claims 1  to  7 , which is conjugated to a polypeptide, a lipid, or a polymer. 
     
     
         9 . The oligosaccharide subunit of  claim 8 , wherein the polypeptide is albumin. 
     
     
         10 . The oligosaccharide subunit of  claim 9 , wherein the polypeptide is bovine serum albumin. 
     
     
         11 . A method of evaluating the binding characteristics of an anti- Pseudomonas aeruginosa  Psl antibody or antigen-binding fragment thereof to  Pseudomonas aeruginosa  Psl epitope, comprising
 (a) contacting the antibody with an oligosaccharide subunit of any one of  claims 1  to  10  under conditions that allow the antibody to bind the oligosaccharide subunit; and   (b) detecting the presence of a complex of the antibody and the oligosaccharide subunit.   
     
     
         12 . The method of  claim 11 , wherein the complex is detect by an ELISA assay. 
     
     
         13 . The method of  claim 11  or  claim 12 , further comprising:
 (c) classifying the antibody as binding to a class II epitope if it binds to each of the tetrasaccharide, pentasaccharide, hexasaccharide, and decasaccharide of  claim 5 ; and 
 (d) classifying the antibody as binding to a class III epitope if it binds to the hexasaccharide or decasaccharide of  claim 5 , but does not bind to the tetrasaccharide or the pentasaccharide of  claim 5 . 
 
     
     
         14 . A method of screening for antibodies which bind to  Pseudomonas aeruginosa  Psl, comprising:
 (a) contacting an antibody or antibody fragment library with an oligosaccharide subunit of any one of  claims 1  to  10  under conditions that allow Psl-specific antibodies or fragments thereof to bind the oligosaccharide subunit;   (b) detecting the presence of a complex of the antibody and the oligosaccharide subunit; and   (c) selecting antibodies which bind to the oligosaccharide subunit.   
     
     
         15 . The method of  claim 14 , wherein the complex is detect by an ELISA assay. 
     
     
         16 . A composition comprising the oligosaccharide subunit of any one of  claims 1  to  10 .