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Infection and Immunity, May 2001, p. 2972-2979, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.2972-2979.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Masae Kuboniwa,1,2 Atsuo Amano,3,4,* Satoshi Shizukuishi,2 Ichiro Nakagawa,1 and Shigeyuki Hamada1

Departments of Oral Microbiology,1 Preventive Dentistry,2 and Oral Science Methodology3 and Division of Special Care Dentistry,4 Osaka University Graduate School of Dentistry, Suita-Osaka, Japan

Received 3 November 2000/Returned for modification 28 December 2000/Accepted 7 February 2001

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 (KGPcd) in hemoglobin binding by P. gingivalis, using a specific immunoglobulin G (IgG) elicited by the administration of plasmid DNA encoding KGPcd or the catalytic domain of Arg-gingipain (RGPcd). The pSeq2A/kgpcd and pSeq2B/rgpcd plasmids were constructed by the ligation of kgpcd and rgpcd DNA fragments, respectively. Female BALB/c mice were immunized with each of these plasmids. pSeq2A/kgpcd elicited a strong response to recombinant KGPcd (rKGPcd), as well as to comparably produced rRGPcd-reactive antibodies. The serum antibodies elicited by pSecTag2B/rgpcd also cross-reacted with rKGPcd as well as rRGPcd. Anti-KGPcd IgG significantly inhibited hemoglobin binding by P. gingivalis. Furthermore, the inhibition of hemoglobin binding was markedly enhanced by a combination of anti-KGPcd and anti-fimbriae. Anti-RGPcd IgG showed a negligible inhibitory effect, while both anti-KGPcd and anti-RGPcd 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/kgpcd and pSeq2B/rgpcd prevented inflammatory responses and prolonged the survival rate of immunized mice by 43 and 27%, respectively. These results suggest that KGPcd acts as a hemoglobin-binding protein and can also be useful as an immunogen inducing a protective response to P. gingivalis infection.

* Corresponding author. Mailing address: Department of Oral Science Methodology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan. Phone: 81-6-6879-2283. Fax: 81-6-6879-2976. E-mail: amanoa{at}dent.osaka-u.ac.jp.

Infection and Immunity, May 2001, p. 2972-2979, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.2972-2979.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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