Fig 1.
A) HPLC-SEC profiles (fluorescence emission detection) of Vi-CRM197 (red), Vi-DT (orange) and Vi-TT (green). TSK gel 6000–5000 PW columns, NaCl 0.1 M NaH2PO4 0.1 M ACN 5%, pH 7.2; flow 0.5 mL/min; Vtot 49.004 min; V0 24.382 min. B). HPLC-SEC profiles of fVi-CRM197 (red), fVi-DT (orange), fVi-TT (green). TSK gel 3000 PWxl column; NaCl 0.1 M NaH2PO4 0.1 M ACN 5%, pH 7.2; flow rate 0.5 mL/min; Vtot 23.326 min, V0 10.663 min.
Table 1.
Characterization of full-length and fragmented Vi conjugates by using different carrier proteins.
Fig 2.
Influence of carrier protein on the immunogenicity of Vi conjugates in mice.
Ten weeks old CD1 mice were immunized at days 0 and 35 with 1 μg Vi/dose. A) anti-Vi IgG ELISA titres and B) anti-carrier IgG ELISA titres. Bars represent the geometric mean ELISA units of the group in log scale, individual animals are represented by the scatter plots.
Fig 3.
Influence of saccharide to protein ratio and conjugate size on: anti-Vi IgG response induced in mice by Vi-CRM197 (A and B respectively) and fVi-CRM197 conjugates (C); anti-CRM197 IgG response induced in mice by fVi-CRM197 conjugates (D). Ten weeks old CD1 mice were immunized at days 0 and 28 or 35 at 1 μg (A, B) and 1 or 8 μg (C) Vi/dose. Bars represent the geometric mean ELISA units of the group in log scale, individual animals are represented by the scatter plots. HMW and LMW indicate high molecular weight and low molecular weight conjugates.
Table 2.
Full-length and fragmented Vi-CRM197 conjugates differing for saccharide to CRM197 ratio or size.
Fig 4.
HPLC-SEC profiles (fluorescence emission detection) on TSK gel 3000 PWxl column (NaH2PO4 100 mM, NaCl 100 mM, 5% CH3CN pH 7.2; 0.5mL/min) of fVi-CRM197 conjugates 1 (pink), 2 (black), 3 (red) and 4 (blue) (Table 2).
Fig 5.
Conjugation scheme for the synthesis of A) fVi-CRM197 conjugates differing for the length of the spacer molecule introduced on CRM197 before conjugation to fVi; B) fVi(DMT-MM)CRMADH; C) fVi(ADH)-CRM197; D) fViADHN3-CRMalkyne; E) fVis(ADH)-CRM197; F) fVisSH-CRMSBAP (Table 3).
Table 3.
Characteristics of the conjugates obtained using different conjugation strategies.
Fig 6.
Influence of conjugation chemistry (Table 3) on the immunogenicity of fVi-CRM197 conjugates in mice: anti-Vi IgG ELISA titres.
Five weeks old CD1 mice were immunized at days 0 and 28 at 1 μg Vi/dose in two separate studies (A and B). Bars represent the geometric mean ELISA units of the group in log scale, individual animals are represented by the scatter plots.