Methods for conjugation of oligosaccharides or polysaccharides to protein carriers through oxime linkages via 3-deoxy-d-manno-octulsonic acid

Abstract

Methods for preparing an oligosaccharide—protein carrier immunogenic conjugate or a polysaccharide—protein carrier immunogenic conjugate. The methods include obtaining an oligosaccharide or polysaccharide having a KDO moiety located at the terminal reducing end of the oligosaccharide or polysaccharide that includes a carbonyl functional group; and reacting the carbonyl functional group of the KDO moiety with an aminooxylated protein carrier molecule resulting in a conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier.

We claim: 
     
         1 . A method for preparing an oligosaccharide—protein carrier immunogenic conjugate or polysaccharide—protein carrier immunogenic conjugate, comprising:
 obtaining an oligosaccharide or polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducing end of the oligosaccharide or polysaccharide that includes a carbonyl functional group; and 
 reacting the carbonyl functional group of the anhydro 3-deoxy-D-manno-octulsonic acid moiety with an aminooxylated protein carrier molecule resulting in an oligosaccharide—protein carrier immunogenic conjugate or polysaccharide—protein carrier immunogenic conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier. 
 
     
     
         2 . The method of  claim 1 , wherein the oligosaccharide or polysaccharide is obtained from  Haemophilus ducreyi, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Vibrio cholere, Shigella  sp.,  Haemophilus influenza , or a mixture thereof. 
     
     
         3 . The method of  claim 1 , wherein the oligosaccharide or polysaccharide is obtained from at least one bacteria containing a lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid moiety. 
     
     
         4 . The method of  claim 1 , wherein obtaining the oligosaccharide or polysaccharide comprises:
 isolating a lipopolysaccharide from at least one type of bacteria, wherein the lipopolysaccharide includes a Lipid A domain and at least one polysaccharide or oligosaccharide domain, the Lipid A domain and the polysaccharide or oligosaccharide domain being joined together by 3-deoxy-D-manno-octulsonic acid; and   cleaving the Lipid A from the polysaccharide or oligosaccharide domain such that the 3-deoxy-D-manno-octulsonic acid remains linked to the polysaccharide or oligosaccharide domain.   
     
     
         5 . The method of  claim 4 , wherein cleaving the Lipid A from the polysaccharide or oligosaccharide domain comprises acid hydrolyzing the lipopolysaccharide under conditions sufficient for severing a glycosidic bond between the 3-deoxy-D-manno-octulsonic acid and the Lipid A domain. 
     
     
         6 . The method of  claim 4 , wherein cleaving the lipid A from the polysaccharide or oligosaccharide domain comprises treating the lipopolysaccharide with acetic acid. 
     
     
         7 . The method of  claim 1 , wherein the carbonyl functional group is a ketone. 
     
     
         8 . The method of  claim 1 , wherein the mol ratio of the carbonyl functional group on the oligosaccharide or polysaccharide:aminooxy on the protein carrier ranges from about 0.3:1 to about 1:3. 
     
     
         9 . The method of  claim 1  wherein the aminooxylated protein carrier is prepared by treating a protein with at least one agent that introduces at least one aminooxy functional group onto the protein. 
     
     
         10 . The method of  claim 9  wherein the aminooxy-introducing agent is selected from aminoooxy-alkyl-thiol and aminoooxy-aryl-thiol. 
     
     
         11 . The method of  claim 9 , further comprising treating the protein with a treatment agent that introduces at least one thiol-reactive group onto the protein prior to treating the protein with the aminooxy-introducing agent. 
     
     
         12 . The method of  claim 11 , wherein the thiol-reactive group is a haloacetamido moiety. 
     
     
         13 . The method of  claim 1 , wherein the oligosaccharide or polysaccharide is obtained from  Bordetella bronchiseptica, Bordetella  parapertussis, or  Bordetella pertussis.    
     
     
         14 . The method of  claim 1 , wherein the oligosaccharide or polysaccharide is obtained from at least one bacteria containing a lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid moiety phosphorylated at position C4 on the 3-deoxy-D-manno-octulsonic acid moiety. 
     
     
         15 . The method of  claim 1 , wherein the oligosaccharide or polysaccharide is obtained from  Shigella flexneri.    
     
     
         16 . A method for preparing an oligosaccharide—protein carrier immunogenic conjugate or polysaccharide—protein carrier immunogenic conjugate, comprising:
 obtaining an oligosaccharide or polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducing end of the oligosaccharide or polysaccharide; 
 reacting the anhydro 3-deoxy-D-manno-octulsonic acid moiety of the oligosaccharide or polysaccharide with a heterobifunctional compound that includes at least one aminooxy group; and then 
 reacting the oligosaccharide or polysaccharide with a protein carrier resulting in an oligosaccharide—protein carrier immunogenic conjugate or polysaccharide—protein carrier immunogenic conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier.