ORAL VACCINATION AGAINST TETANUS - COMPARISON OF THE IMMUNOGENICITIESOF SALMONELLA STRAINS EXPRESSING FRAGMENT-C FROM THE NIRB AND HTRA PROMOTERS

We have found the in vivo-regulated nirB promoter (P-nirB) to be effec tive for directing expression of a number of antigens in salmonella in vivo. We wished to determine if other in vivo-regulated promoters hav e utility for antigen expression in salmonella and to compare the effe ctiveness of these promoters with that of P-nirB. To this end, we have devised a scheme that allows the promoter element of the P-nirB-fragm ent C plasmid pTETnir15 to be swapped with other promoters of interest . We demonstrate the usefulness of this system by replacing P-nirB wit h P-htrA to create plasmid pTEThtrA1. htrA is a stress response gene t hat is required for virulence of salmonella in mice and survival withi n macrophages. Expression of fragment C in Salmonella typhimurium BRD5 09 (aroA aroD) harboring pTEThtrA1 (strain BRD937) correlated with gro wth temperature in vitro. A comparison was made of the immune response s to fragment C elicited in mice immunized orally with BRD937 or BRD83 7 (BRD509/pTETnir15) or subcutaneously with purified fragment C plus a lhydrogel. High levels of anti-fragment C antibodies that persisted fo r at least 12 weeks were present in all groups of mice. Vaccination wi th BRD937 was the most effective means of immunization: the serum immu noglobulin G (IgG), IgA, and IgM anti-fragment C titers were higher in the BRD937-immunized mice throughout the duration of the study than i n mice in the other groups. The kinetics of the serum anti-fragment C responses were different in different groups. The response was most ra pid in the BRD937 group, with the titers almost at peak levels at 2 we eks postimmunization. Only the mice immunized with BRD937 or BRD837 de veloped an intestinal IgA response to fragment C. Again, the response was superior in the BRD937 group. The peak of the intestinal response was delayed with respect to the serum response. Analysis of the Ige su btype response to fragment C revealed a dominant IgG2a response in the salmonella-immunized mice, indicating a type 1 helper T-cell response to fragment C, whereas the major subtype in the group parenterally im munized with fragment C plus alhydrogel was IgG1. The IgG1/IgG2a ratio was much higher in sera of BRD937-immunized mice than in sera of BRD8 47-immunized mice. At 15 to 20,weeks after immunization, the mice immu nized with BRD937 or BRD817 were solidly immune to tetanus toxin and s almonella. The immune responses to fragment C seen in mice immunized w ith BRD937 are the strongest,ve have observed and indicate that the ht rA promoter may be very useful for expressing foreign antigens in salm onella vaccine strains.