Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Abstract

Background: Gardnerella vaginalis is a bacterial vaginosis-associated vaginal bacterium, which produces toxin vaginolysin (VLY). VLY is a pore-forming toxin suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and emergence of antibiotic-resistant bacterial species demonstrate the need for development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve properties of scFvs the monospecific dimeric scFvs were generated by a genetic fusion of two anti-VLY scFv molecules connected with the alpha-helix-forming peptide linker.

Results: N-terminally hexahistidine-tagged dimeric scFvs were constructed, produced in Escherichia coli and purified using metal-chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by the in vitro hemolytic assay. Circulating half-life of purified scFvs in blood plasma of mice was accessed using ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life in comparison with parental monomeric scFv.

Conclusions: The protein obtained by a genetic fusion of two anti-VLY scFvs into dimeric molecule exhibited increased properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by bacteria G. vaginalis