Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection

Lys-gingipain (KGP), a lysine-specific cysteine proteinase, is one of the m ajor virulence factors of Porphyromonas gingivalis. Here we examined the in volvement of the catalytic domain of KGP (KGP(cd)) in hemoglobin binding by P. gingivalis, using a specific immunoglobulin G (IgG) elicited by the adm inistration 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 co nstructed by the ligation of kgp(cd) and rgp(cd) DNA fragments, respectivel y. Female BALB/c mite were immunized with each of these plasmids, pSeq2A/kg p(cd) elicited a strong response to recombinant KGP(cd) (rKGP(cd)), as well as to comparably produced rRGP(cd)-reactive antibodies. The serum antibodi es elicited by pSecTag2B/rgp(cd) also cross-reacted with rKGP(cd) as well a s rRGP(cd). Anti-KGP(cd) IgG significantly inhibited hemoglobin binding by P, gingivalis, Furthermore, the inhibition of hemoglobin binding was marked ly 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 an ti-RGP(cd) IgGs showed significant inhibitory effects on Lys- and Arg- spec ific proteolytic activities and on the growth of P, gingivalis under iron-r estricted conditions where supplemented hemoglobin was the sole iron source . Immunized mice were challenged by intraperitoneal inoculation with P. gin givalis. All nonimmunized mice died within 72 h; however, vaccination with pSeq2A/kgp(cd) and pSeq2B/rgp(cd) prevented inflammatory responses and prol onged the survival rate of immunized mice by 43 and 27%, respectively. Thes e 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. gingiv alis infection.