Specific Antibodies to Porphyromonas gingivalis Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection

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Abstract
Lys-gingipain (KGP), a lysine-specific cysteine proteinase, is one of the major virulence factors of Porphyromonas gingivalis . Here we examined the involvement of the catalytic domain of KGP (KGP cd ) in hemoglobin binding by P. gingivalis , using a specific immunoglobulin G (IgG) elicited by the administration of plasmid DNA encoding KGP cd or the catalytic domain of Arg-gingipain (RGP cd ). The pSeq2A/ kgp cd and pSeq2B/ rgp cd plasmids were constructed by the ligation of kgp cd and rgp cd DNA fragments, respectively. Female BALB/c mice were immunized with each of these plasmids. pSeq2A/ kgp cd elicited a strong response to recombinant KGP cd (rKGP cd ), as well as to comparably produced rRGP cd -reactive antibodies. The serum antibodies elicited by pSecTag2B/ rgp cd also cross-reacted with rKGP cd as well as rRGP cd . Anti-KGP cd IgG significantly inhibited hemoglobin binding by P. gingivalis . Furthermore, the inhibition of hemoglobin binding was markedly enhanced by a combination of anti-KGP cd and anti-fimbriae. Anti-RGP cd IgG showed a negligible inhibitory effect, while both anti-KGP cd and anti-RGP cd IgGs showed significant inhibitory effects on Lys- and Arg-specific proteolytic activities and on the growth of P. gingivalis under iron-restricted conditions where supplemented hemoglobin was the sole iron source. Immunized mice were challenged by intraperitoneal inoculation with P. gingivalis . All nonimmunized mice died within 72 h; however, vaccination with pSeq2A/ kgp cd and pSeq2B/ rgp cd prevented inflammatory responses and prolonged the survival rate of immunized mice by 43 and 27%, respectively. These results suggest that KGP cd acts as a hemoglobin-binding protein and can also be useful as an immunogen inducing a protective response to P. gingivalis infection.