Chapter 16
Exploitation of Capsule Polymerases for Enzymatic
Synthesis of Polysaccharide Antigens Used
in Glycoconjugate Vaccines
Christa Litschko, Insa Budde, Monika Berger, and Timm Fiebig
Abstract
The exploitation of recombinant enzymes for the synthesis of complex carbohydrates is getting increasing
attention. Unfortunately, the analysis of the resulting products often requires advanced methods like
nuclear magnetic resonance spectroscopy and mass spectrometry. Here, we use the capsule polymerases
Cps4B and Cps11D from Actinobacillus pleuropneumoniae serotypes 4 and 11, respectively, as examples for
the in vitro synthesis of capsule polymers similar to those used in glycoconjugate vaccine formulations. We
demonstrate how substrate turnover in an enzymatic reaction can be analyzed by HPLC-based anion
exchange chromatography and provide the protocol for separation and detection of UV-active polymer.
Moreover, we describe how UV-inactive polymer can be separated and visualized using polyacrylamide gel
electrophoresis followed by combined alcian blue–silver staining.
Key words In vitro vaccine production, Capsule polymerase, Capsular polysaccharide, Enzymatic
synthesis, Glycoconjugate vaccine, HPLC, PAGE, Alcian blue–silver staining, Actinobacillus
pleuropneumoniae
1 Introduction
Capsular polysaccharides belong to the main virulence factors of
bacterial pathogens by providing protection against host immune
defense mechanisms [1]. However, since they represent the outermost layer of the cell and thus the interface to the host immune
system, they can also be used as antigens in glycoconjugate vaccine
formulations. Glycoconjugate vaccines have been tremendously
effective in combatting diseases caused by various bacterial pathogens [2]. Through coupling of the saccharide antigen to a carrier
protein, a T-cell dependent immune response is achieved, including
immunologic memory, immunoglobulin M to immunoglobulin G
isotype switching, and protection of infants [3]. The conventional
production of glycoconjugate vaccines includes the purification of
the capsule polymer from pathogen cultures (Fig. 1). This
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_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
313
Exploitation of Capsule Polymerases for Enzymatic
Synthesis of Polysaccharide Antigens Used
in Glycoconjugate Vaccines
Christa Litschko, Insa Budde, Monika Berger, and Timm Fiebig
Abstract
The exploitation of recombinant enzymes for the synthesis of complex carbohydrates is getting increasing
attention. Unfortunately, the analysis of the resulting products often requires advanced methods like
nuclear magnetic resonance spectroscopy and mass spectrometry. Here, we use the capsule polymerases
Cps4B and Cps11D from Actinobacillus pleuropneumoniae serotypes 4 and 11, respectively, as examples for
the in vitro synthesis of capsule polymers similar to those used in glycoconjugate vaccine formulations. We
demonstrate how substrate turnover in an enzymatic reaction can be analyzed by HPLC-based anion
exchange chromatography and provide the protocol for separation and detection of UV-active polymer.
Moreover, we describe how UV-inactive polymer can be separated and visualized using polyacrylamide gel
electrophoresis followed by combined alcian blue–silver staining.
Key words In vitro vaccine production, Capsule polymerase, Capsular polysaccharide, Enzymatic
synthesis, Glycoconjugate vaccine, HPLC, PAGE, Alcian blue–silver staining, Actinobacillus
pleuropneumoniae
1 Introduction
Capsular polysaccharides belong to the main virulence factors of
bacterial pathogens by providing protection against host immune
defense mechanisms [1]. However, since they represent the outermost layer of the cell and thus the interface to the host immune
system, they can also be used as antigens in glycoconjugate vaccine
formulations. Glycoconjugate vaccines have been tremendously
effective in combatting diseases caused by various bacterial pathogens [2]. Through coupling of the saccharide antigen to a carrier
protein, a T-cell dependent immune response is achieved, including
immunologic memory, immunoglobulin M to immunoglobulin G
isotype switching, and protection of infants [3]. The conventional
production of glycoconjugate vaccines includes the purification of
the capsule polymer from pathogen cultures (Fig. 1). This
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_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
313