RgpA-Kgp peptide-based immunogens provide protection against Porphyromonasgingivalis challenge in a murine lesion model

Porphyromonas gingivalis, a gram-negative bacterium, has been linked to the onset and progression of periodontit is, a chronic inflammatory disease of the supporting tissues of the teeth. A major virulence factor of P. gingiv alis is an extracellular complex of Arg- and Lys-specific proteinases and a dhesins designated the RgpA-Kgp complex (formerly the PrtR-PrtK complex). I n this study we show that the RgpA-Kgp complex, when used as an immunogen w ith incomplete Freund adjuvant (IFA), protects against challenge with invas ive and noninvasive strains of P. gingivalis in the murine lesion model. We identified a variety of peptide vaccine candidates from the RgpA and Kgp p olyprotein sequences that involved the putative active site histidine of bo th proteinases and Eve repeat motifs in the adhesin domains of both polypro teins implicated in aggregation and binding to host substrates, designated adhesin binding motif (ABM) peptides. These peptides were synthesized using standard, solid-phase protocols for 9-fluorenylmethoxy carbonyl chemistry with S-acetylmercaptoacetic acid (SAMA) as the N-terminal residue. The SAMA -peptides were then conjugated to diphtheria toroid and used with IPA to im munize BALB/c mice. Both active-site peptides and three of the five ABM pep tides gave protection (P < 0.005) against challenge with P. gingivalis in t he murine lesion model. The three ABM peptide sequences that conferred prot ection exist within a 100-residue span in the RgpA44 and Kgp39 adhesins of the RgpA-Kgp complex. Protective anti-RgpA-Kgp complex: mouse antisera reco gnized the RgpA27, Kgp39, and RgpA44 adhesins in an immunoblot. Epitope map ping of the RgpA27 adhesin using the protective anti-RgpA-Kgp antisera iden tified a major protective epitope that mapped immediately N terminal to one of the protective ABM peptides in the 100-residue span in RgpA44 and Kgp39 . This identified protective epitope contains clusters of basic residues sp atially surrounded by hydrophobic amino acids, a finding which is character istic of a heparin binding motif.