CLONING OF THE AMINO TERMINAL NUCLEOTIDES OF THE ANTIGEN-I/II OF STREPTOCOCCUS-SOBRINUS AND THE IMMUNE-RESPONSES TO THE CORRESPONDING SYNTHETIC PEPTIDES

A portion of the antigen I/II (spaA, B, Pl) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage-lambda-GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of ]H-3[-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation. It thus appears that there is a region in the antigen I/II protein, between residues 211 and 327 for Strep. sobrinus and between residues 197 and 330 for Strep. mutans molecule, that is accessible to antibodies in vivo; it is in an amphipathic helix of the native molecule, which may have been conserved through evolution. Synthetic peptides generated to one or more of these regions by recombinant DNA technology may be the basis for a vaccine against dental caries.