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Published on: October 13, 2020
Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing
Maria Michelina Raso1, Oscar Vassallo1,2, Francesca Micoli1
1GSK Vaccines Institute for Global Health (GVGH) S.r.l., Via Fiorentina 1, 53100 Siena, Italy.
Insights
Two methods were optimized to quantify Shigella flexneri serotype 6 O-antigen, a key vaccine component. These improved methods enhance vaccine quality control and support the development of Shigella vaccines against diarrheal diseases.
Area of Science:
- Vaccinology
- Microbiology
- Analytical Chemistry
Background:
- Shigella causes significant global diarrheal disease, particularly in children.
- Antimicrobial resistance necessitates the development of effective Shigella vaccines.
- Shigella flexneri serotype 6 is a target for broad-protection vaccines, with its O-antigen (OAg) being a critical component.
Purpose of the Study:
- To optimize and characterize two methods for quantifying Shigella flexneri 6 O-antigen.
- To improve the quality control of Shigella OAg-based vaccines.
- To facilitate the development and release of Shigella vaccines.
Main Methods:
- Optimization of a Dische colorimetric method for detecting methyl-pentoses (rhamnose) in S. flexneri 6 OAg.
- Development of a High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) method for detecting specific dimers (GalA-GalN) from S. flexneri 6 OAg hydrolysis.
- Characterization of the performance and sensitivity of both optimized methods.
Main Results:
- The optimized Dische method showed a tenfold increase in sensitivity for detecting rhamnose.
- The HPAEC-PAD method provided specific detection of a dimer derived from S. flexneri 6 OAg.
- Both methods demonstrated utility for characterizing S. flexneri 6 OAg.
Conclusions:
- Optimized colorimetric and HPAEC-PAD methods enhance the quantification of Shigella flexneri 6 OAg.
- These analytical advancements support the quality assessment of Shigella OAg-based vaccines.
- The methods have potential applications for quantifying other polysaccharides.
Abstract:
Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.
