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Antimicrobial Agents and Chemotherapy, September 2006, p. 3090-3097, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01435-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Inhibition of Fungal ß-1,3-Glucan Synthase and Cell Growth by HM-1 Killer Toxin Single-Chain Anti-Idiotypic Antibodies

Dakshnamurthy Selvakumar,1 Masahiko Miyamoto,1 Yasuhiro Furuichi,2 and Tadazumi Komiyama1*

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan,1 GeneCare Research Institute Co. Ltd., 200 Kajiwara, Kamakura 247-0063, Japan2

Received 8 November 2005/ Returned for modification 23 December 2005/ Accepted 17 June 2006

Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis. scFv antibodies were fungicidal at concentrations of 1.56 to 12.5 µg/ml in vitro against four Candida species. The scFv antibodies exerted a strong candidacidal activity in vitro, with 50% inhibitory concentration (IC50) values ranging from 7.3 x 10–8 to 16.0 x 10–8 M, and were neutralized by adsorption with nMAb-KT. Furthermore, all scFv antibodies effectively inhibited fungal ß-1,3-glucan synthase activity in vitro, with IC50 values ranging from 2.0 x 10–8 to 22.7 x 10–8 M, values which almost coincide with the values that are inhibitory to the growth of fungal cells. Binding assays showed that the scFv antibodies specifically bind to nMAb-KT, and this binding pattern was confirmed by surface plasmon resonance analysis. The binding ability was further demonstrated by the competition observed between scFv antibodies and HM-1 to bind nMAb-KT. To the best of our knowledge, this is the first study to show that an antifungal anti-idiotypic antibody, in the form of recombinant scFv, potentially inhibits ß-1,3-glucan synthase activity.

* Corresponding author. Mailing address: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan. Phone: 81-250-25-5273. Fax: 81-250-25-5021. E-mail: tkomiyam{at}niigata-pharm.ac.jp.

Antimicrobial Agents and Chemotherapy, September 2006, p. 3090-3097, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01435-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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