Chapter 14
O-Antigen Extraction, Purification, and Chemical
Conjugation to a Carrier Protein
Francesca Micoli, Carlo Giannelli, and Roberta Di Benedetto
Abstract
A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more
vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate
vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are
making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules.
Here, we report a simplified method for O-antigen extraction and purification that avoids the step of
lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier
protein is described, together with analytical methods for intermediates and final conjugate
characterization.
Key words O-antigen, Polysaccharide, Glycoconjugation, Vaccine, Purification, Salmonella
1 Introduction
Surface polysaccharides from bacteria have been used for many
years in vaccine applications, being both essential virulence factors
and targets for protective antibodies. Covalent conjugation to an
appropriate carrier protein was discovered to be an important
means to increase the immunogenicity of polysaccharides
[1, 2]. Glycoconjugates provide T cell-dependent immunogenicity
against the saccharide hapten. With the involvement of T cells,
immunological memory is invoked, and affinity maturation and
isotype switching occur. Differently from polysaccharides, glycoconjugate vaccines are effective in young infants [3, 4].
Licensed glycoconjugate vaccines are produced by chemical
conjugation of capsular polysaccharides to prevent meningitis
caused by meningococcus, pneumococcus and Haemophilus influenzae type b. However, other classes of carbohydrates, including
the O-antigen portion of lipopolysaccharide (LPS) molecules in
gram-negative bacteria, represent attractive targets for glycoconjugates. O-antigen-based conjugate vaccines have been proposed for
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
267
O-Antigen Extraction, Purification, and Chemical
Conjugation to a Carrier Protein
Francesca Micoli, Carlo Giannelli, and Roberta Di Benedetto
Abstract
A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more
vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate
vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are
making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules.
Here, we report a simplified method for O-antigen extraction and purification that avoids the step of
lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier
protein is described, together with analytical methods for intermediates and final conjugate
characterization.
Key words O-antigen, Polysaccharide, Glycoconjugation, Vaccine, Purification, Salmonella
1 Introduction
Surface polysaccharides from bacteria have been used for many
years in vaccine applications, being both essential virulence factors
and targets for protective antibodies. Covalent conjugation to an
appropriate carrier protein was discovered to be an important
means to increase the immunogenicity of polysaccharides
[1, 2]. Glycoconjugates provide T cell-dependent immunogenicity
against the saccharide hapten. With the involvement of T cells,
immunological memory is invoked, and affinity maturation and
isotype switching occur. Differently from polysaccharides, glycoconjugate vaccines are effective in young infants [3, 4].
Licensed glycoconjugate vaccines are produced by chemical
conjugation of capsular polysaccharides to prevent meningitis
caused by meningococcus, pneumococcus and Haemophilus influenzae type b. However, other classes of carbohydrates, including
the O-antigen portion of lipopolysaccharide (LPS) molecules in
gram-negative bacteria, represent attractive targets for glycoconjugates. O-antigen-based conjugate vaccines have been proposed for
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
267