Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.
Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.
Neil
N
Ravenscroft
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Micha A
MA
Haeuptle
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Michael
M
Kowarik
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Fabiana S
FS
Fernandez
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Paula
P
Carranza
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Andreas
A
Brunner
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Michael
M
Steffen
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Michael
M
Wetter
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Sacha
S
Keller
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.
Corina
C
Ruch
GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland michael.wacker@glycovaxyn.com.
Michael
M
Wacker
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D016052:Protozoan Vaccines / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D012762:Shigella dysenteriae / Q000276:immunology*; D018074:Vaccines, Conjugate / Q000276:immunology
D001426:Bacterial Proteins; D004731:Endotoxins; D006023:Glycoproteins; D015800:Protozoan Proteins; D016052:Protozoan Vaccines; D018074:Vaccines, Conjugate; D016695:Glycosyltransferases
E. coli glycosylation; O-polysaccharide; Shigella dysenteriae type 1; biosynthetic glycoconjugate vaccine; vaccine development