EXPRESSION AND IMMUNOGENICITY OF PERTUSSIS TOXIN S1 SUBUNIT-TETANUS TOXIN FRAGMENT-C FUSIONS IN SALMONELLA-TYPHI VACCINE STRAIN CVD-908

Salmonella typhi vaccine strain CVD 908 can deliver heterologous antig ens to the host immune system following mucosal immunization. Stable e xpression of foreign proteins in Salmonella cells often requires antig en-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antige ns to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis t oxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxy l terminus of fragment C mere expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal a ntibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these re combinant plasmids. All fusion proteins elicited serum antibody respon ses to fragment C following intranasal immunization, whereas oral inoc ulation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less e ffective than S1 fused to the carboxyl terminus in generating anti-fra gment C antibodies. CVD 908 expressing truncated SI fused to the carbo xyl terminus of fragment C elicited neutralizing serum pertussis antit oxin following intranasal immunization of mice.