Methods for conjugation of oligosaccharides or polysaccharides to protein carriers through oxime linkages via 3-deoxy-D-manno-octulsonic acid
Inventors
Kubler-Kielb, Joanna • Pozsgay, Vince • Lagergard, Teresa • Ben-Menachem, Gil • Schneerson, Rachel • Vinogradov, Evguenii • Ginzberg, Ariel
Assignees
National Research Council of Canada • US Department of Health and Human Services
Publication Number
US-8795680-B2
Publication Date
2014-08-05
Expiration Date
2027-07-18
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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.
Core Innovation
The invention disclosed are methods for preparing immunogenic conjugates comprising oligosaccharides or polysaccharides conjugated to protein carriers via an oxime linkage formed through the 3-deoxy-D-manno-octulsonic acid (KDO) moiety located at the terminal reducing end of the oligosaccharide or polysaccharide. The methods involve obtaining an oligosaccharide or polysaccharide containing an anhydro 3-deoxy-D-manno-octulsonic acid moiety with a carbonyl functional group and reacting this with an aminooxylated protein carrier to create a conjugate with a covalent oxime bond connecting the saccharide to the protein carrier.
This approach solves the problem of preserving the native structure of oligosaccharides or polysaccharides from Gram-negative bacteria such as Bordetella spp., Haemophilus ducreyi, and Shigella flexneri, where traditional methods of conjugation may oxidize or modify saccharide residues. The invention maintains the integrity of the external non-reducing end epitopes critical for immunogenicity by conjugating via the KDO moiety without oxidizing the oligosaccharide chain, allowing the conjugate to be recognized by specific antisera and to induce immune responses.
The oxime linkage formed is specific and stable under mild aqueous conditions (pH 5 to 7) at ambient temperature, which avoids protein denaturation and allows for high yields with controlled loading (5 to 20 saccharide chains per protein). The use of aminooxylated protein carriers functionalized through heterobifunctional linkers enables selective and efficient conjugation. Such conjugates demonstrate immunogenicity and specificity in animal models, indicating their potential for developing vaccines against bacterial infections caused by pathogens including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus ducreyi, Vibrio cholerae, Haemophilus influenza, and Shigella species.
Claims Coverage
The patent discloses 4 main inventive features centered around immunogenic conjugates comprising oligosaccharides or polysaccharides conjugated to carrier proteins via oxime bonds formed through a 3-deoxy-D-manno-octulsonic acid moiety, pharmaceutical compositions containing such conjugates, and methods of eliciting an immune response using these conjugates.
Immunogenic conjugates formed via oxime linkage through anhydro 3-deoxy-D-manno-octulsonic acid moiety
The conjugate comprises a structure Pr-Sp-O—N═C(COOH)-anh-KDO—OS, where Pr is a carrier protein, Sp is an optional spacer, anh-KDO is the anhydro 3-deoxy-D-manno-octulsonic acid moiety, and OS is an oligosaccharide or polysaccharide residue derived from cleavage of Lipid A from lipopolysaccharide. The lipopolysaccharide is obtained from specified Gram-negative bacteria including Haemophilus ducreyi, Bordetella spp., Vibrio cholerae, and Haemophilus influenzae.
Specific composition of the oligosaccharide or polysaccharide
The oligosaccharide or polysaccharide includes an O-antigen chain and a core oligosaccharide, with specific examples comprising pentasaccharides identified in Bordetella bronchiseptica and Bordetella parapertussis, or specific oligosaccharide compositions from Haemophilus ducreyi.
Pharmaceutical compositions incorporating the conjugates
Pharmaceutical compositions comprising the described immunogenic conjugates with pharmaceutically acceptable carriers suitable for administration are claimed, facilitating their use in therapeutic or prophylactic contexts.
Method of eliciting an immune response
A method of eliciting an immune response in a subject by administering the immunogenic conjugate comprising the oligosaccharide or polysaccharide bound via an oxime bond through the 3-deoxy-D-manno-octulsonic acid moiety to a carrier protein, thereby triggering an immune response against the bacterial antigen.
The claims cover immunogenic conjugates of saccharides linked to carrier proteins through a stable oxime bond via the KDO moiety, specific compositions of these saccharides and carriers, pharmaceutical formulations thereof, and methods of immunization using these conjugates, providing a comprehensive protection for the innovative bio-conjugation approach and its applications in vaccine development.
Stated Advantages
Preserves the conserved external non-reducing end epitopes of oligosaccharides or polysaccharides by conjugating via the KDO moiety without oxidizing the saccharide residues, leading to higher immunogenicity.
Forms stable oxime linkages in aqueous solution at ambient temperatures and physiological pH ranges, avoiding protein denaturation and maintaining biological activity.
Allows controlled and efficient conjugation with high yield, introducing about 5 to 20 oligosaccharide chains per carrier protein molecule.
The conjugates induce specific immune responses in animal models, demonstrating potential as effective vaccines against various Gram-negative bacterial pathogens.
Documented Applications
Use as immunogenic compositions or vaccines for prevention and treatment of infections caused by Gram-negative bacteria including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus ducreyi, Vibrio cholerae, Shigella species, and Haemophilus influenza.
Preparation of immunogenic conjugates for eliciting specific antibody responses against bacterial lipopolysaccharide or lipooligosaccharide antigens.
Formulation into pharmaceutical compositions for administration by various mucosal and non-mucosal routes for prophylactic or therapeutic purposes.
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