A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA 2 ) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA 2 was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.Many childhood vaccines, such as the diphtheria-pertussistetanus vaccine, require multiple doses to achieve protection, which has led to reduced coverage of immunization. Live viral or bacterial vectors have been suggested as a solution to reduce the number of doses required for immunization. In addition, live vectors have the ability to induce a mucosal immune response following oral or nasal administration, which is typically difficult to obtain from conventional parenteral immunization. Among these bacteria, S. gordonii is a very attractive vector for childhood vaccines because it is one of the pioneer organisms in the human oral cavity. The organism can be detected in the oral cavity by 6 months of age and remains as a normal inhabitant of the oral mucosa and dental plaque in adults. Therefore, the organism has the potential of providing lifelong protection following a single oral inoculation. In addition, this bacterium is relatively easy to manipulate genetically, and the expression of a number of heterologous antigens has been reported (18,20,22,23,24,30). In the context of a live oral diphtheria-pertussis-tetanus vaccine, Medaglini et al. (24) showed that immunization with recombinant S. gordonii expressing tetanus toxin fragment C on its surface conferred protection from lethal tetanus toxin challenge in mice. In a previous work, we showed that the surface-expressed pertussis toxin S1 fragment (18, 19) and a secreted fusion protein consisting of pertussis toxin S1S3 fragments and filamentous hemagglutinin type I domain (20) produced by S. gordonii were immunogenic. These findings show that tetanus and pertussis antigens can be expressed by S. gordonii and that the antigens were immunogenic. However, the expression of diphtheria antigens in S. gordonii has not yet been reported.Diphtheria toxin (DT) is a secreted 535-amino-acid protein which is proteolytically cleaved into two fragments, A and B (4, 9). The catalytic domain is located on fragment A (amino acids 1 to 193), and the receptor and translocation domains are on fragment B (amino acids 194 to 535). Fragment B is responsible for DT binding to specific...

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