Fig. 6

Analysis of the glycoconjugate vaccine candidate in vitro. a Schematic representation of the glycoconjugate vaccine candidate and its unglycosylated counterpart serving as a negative control; b Estimated biomass of vaccine strains after overnight culture at 28 °C, values plotted are OD_600nm means ± standard deviations of triplicates; c SDS-PAGE followed by western blotting of His-purified G-NetB(10) and unG-NetB expressed in χ7122 pgl from either IPTG-inducible pEXT20 or constitutive pFPV25.1 backbone, percentage in parenthesis indicates sample loading (v/v) per lane; d, e semi-quantitative densitometry analysis of glycan and protein content of G-NetB(10) glycoconjugates (d) and unG-NetB control (e), values plotted are OD-normalised mean intensities ± standard deviations of triplicates (see western blots in Additional file 1: Figure S5 for details), AU, arbitrary units; f Sandwich ELISA on purified glycoconjugate G-NetB(10) and unG-NetB control to relatively quantify the levels of heptasaccharide coupled to G-NetB(10) carrier expressed from the χ7122 pgl strains. g Whole cell ELISA on the CLM24 cedA::pglB glycoengineering strain and χ7122 pgl vaccine strains to relatively quantify the surface display of G-NetB(10) and unG-NetB. IPTG was used to induce NetB expression from pEXT20 vector in the glycoengineering strain, NetB expression from pFPV25.1 in the vaccine strains is instead constitutive. The green dotted line represents a non-specific signal detected when staining CLM24 cedA::pglB + pACYCpglΔpglB (left) and χ7122 wt cells (right) with SBA lectin. Values plotted in (f-g) are background subtracted OD_450nm means ± standard deviations of three biological replicates. Background is set to zero, glycosylation signal of unG-NetB constructs was below background. Panels c-f are different analysis of the same set of triplicates. Statistically significant differences are highlighted with * indicating p < 0.05 (two-tailed t-test). h OCW on CLM24 cedA::pglB glycoengineering strain and χ7122 pgl vaccine strains to visually detect the surface display of G-NetB(10) and unG-NetB. Cell permeabilization was performed to detect NetB antigens localised into the periplasm. IPTG was used to induce NetB expression from pEXT20 vector in the glycoengineering strain, while NetB expression from pFPV25.1 in the vaccine strains is constitutive. Empty wells stained with both primary and secondary antibodies were used as negative controls (− ctrl), while O-antigen negative, WaaL positive E. coli cells expressing pSECpgl were used as positive controls (+ ctrl) for surface display of the heptasaccharide (lipid-linked surface glycan)