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Background   Gardnerella vaginalis  is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by  G. vaginalis . VLY possesses cytolytic activity and is considered as a main virulence factor of  G. vaginalis . Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance.    Results  Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in  E. coli . For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G 4 S) 4  were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by  in vitro  hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent K d  values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells.    Conclusions  Hybridoma-derived scFvs with VLY-binding activity were expressed in  E. coli . Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system.  G. vaginalis  caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by  G. vaginalis .

bmc biotechnology

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis