Figure 4.

Plasma samples from mice immunized with the combination of Vi and CWPS fusion conjugates bind to Vi and promote antibody-mediated killing of Vi-expressing Salmonella strain. (A). ELISA measured the plasma concentrations of anti-Vi IgG. Mice immunized with the combination of the two fusion conjugates had significantly higher plasma concentrations of anti-Vi IgG than mice immunized with the mixture of all antigens (P<0.0001 by Mann-Whitney U). (B) Western blotting analysis revealed that plasma from conjugate-immunized mice binds to Vi-bearing S. typhimurium (C5.507 strain) whereas no binding could be observed with plasma from mixture-immunized mice; no binding could be observed to the non Vi-bearing Salmonella typhimurium strain C5, confirming specificity of the immune response. (C) Sera from mice immunized with the CWPS and Vi fusion conjugates enhanced killing of Vi-expressing Salmonella. Plasma from mice immunized with the two fusion conjugates or with the mixture of all components were used in an killing assay using differentiated human HL-60 cells and strain C5.507. Bacterial growth was observed over a 3-hour period. C5.507 strain incubated with plasma from conjugate-immunized mice demonstrated a 2-log reduction in bacterial CFU, whereas no killing could be observed when plasma from mixture-immunized mice was used. The figure shows one representative result from 3 independent experiments.