Purification and characterization of aShigellaconjugate vaccine, produced by glycoengineeringEscherichia coli

Abstract

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.

Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study

“…Moreover, due to the nonspecific nature of the chemical linkage, the resulting vaccine was heterogeneous, variable from batch to batch, and produced via a relatively low yield. LimmaTech (previously GlycoVaxyn) has developed a novel technology that enables the in vivo biosynthesis of bioconjugate vaccines by directly linking the polysaccharides to the carrier protein using engineered bacterial cells (24). This process allows in vivo conjugation of bacterial polysaccharides to specific consensus sites for N-glycosylation of any carrier protein and removes the requirement for chemical detoxification of LPS and further conjugation processes, in a potentially cost-effective manner.…”

“…The protein component amounts to approximately 50 μg EPA. This material has been characterized extensively using various standard analytical methods, including assays for content, purity, and structure (24). …”

Classical and novel strategies to develop aShigellaglycoconjugate vaccine: from concept to efficacy in human

“…Conjugate characterization and detailed analysis could be achieved by physicochemical methods, including NMR, compatible with quality control to ensure lot-to-lot consistency. 77 Properly engineering of the consensus sequences within a protein carrier could in fine allow ideal control of the O-Ag:carrier loading and attachment site of the polysaccharide component. In the present case, detailed analysis of the bioconjugates demonstrated that although the newly engineered EPA cs mutants could be glycosylated at either site when containing a single N-glycosylation consensus sequence located at two different positions, the use of engineered EPA cs comprising two PglB-compatible consensus sequences resulted only in monoglycosylated SD1 EPA cs conjugates.…”

Shigella Vaccine Development: Finding the Path of Least Resistance

“…As opposed to traditional purification followed by detoxification of LPS and then chemical conjugation to a protein carrier, this candidate vaccine was biosynthesized using recombinant Escherichia coli expressing the Campylobacter jejuni oligosaccharyltrasferase PglB, which transfers the O-antigen repeating unit of S. flexneri 2a LPS to asparagine residues of the periplasmic carrier protein EPA, resulting in stable N-glycosidic linkage (48). Chemical conjugation methods have achieved considerable success but require multistep procedures, resulting in an expensive process that is prone to producing lot-to-lot heterogeneity.…”