Characterization of Shigella GMMA Vaccines

S. sonnei, S. flexneri 2a, and S. Typhimurium GMMA were produced and purified as previously described [6 (link)]. The total protein content was estimated by bicinchoninic acid assay (BCA) using BSA as a reference. The total OAg amount and sugar composition were determined by HPAEC–PAD analysis, after performing acid hydrolysis directly on GMMA. In particular, the OAg amount was quantified based on the detection of repeating unit-specific monosaccharides (2-aminouronic acid for S. sonnei; rhamnose for S. flexneri 2a and S. Typhimurium), as previously described [34 (link),35 (link),36 (link)]. Purity and particle sizes were established by HPLC–SEC MALS, using TSK gel G6000PW + G4000PW columns (Tosoh Bioscience, Tokio, Japan) in series equilibrated in Phosphate Buffer Saline (PBS) [37 (link)]. The OAg extracted was characterized by HPLC–SEC (TSK gel 3000 PWXL column with TSK gel PWXL guard column equilibrated in 0.1 NaCl, 0.1 NaH₂PO₄, 5% CH₃CN, Tosoh Bioscience, Tokio, Japan) with differential refractive index (dRI) detection using dextrans as the standards to estimate the molecular size distribution, as previously described [34 (link),35 (link)]. The ability of each GMMA to stimulate IL-6 release from human PBMC was evaluated by MAT [38 (link)].