title: Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis.
abstract: 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 (G₄S)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 Kd 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.
authors: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania. mildap@ibt.lt
authors: Milda
authors: M
authors: Pleckaityte
authors: Edita
authors: E
authors: Mistiniene
authors: Rita
authors: R
authors: Lasickiene
authors: Gintautas
authors: G
authors: Zvirblis
authors: Aurelija
authors: A
authors: Zvirbliene
mesh_terms: D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000009:adverse effects*; D001427:Bacterial Toxins / Q000009:adverse effects*; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli; D005260:Female; D006194:Gardnerella vaginalis / Q000187:drug effects* / Q000276:immunology; D006367:HeLa Cells; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002479:Inclusion Bodies / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014169:Transformation, Bacterial; D014621:Vagina / Q000187:drug effects / Q000276:immunology / Q000382:microbiology*; D016585:Vaginosis, Bacterial / Q000188:drug therapy* / Q000276:immunology / Q000382:microbiology; D037521:Virulence Factors / Q000009:adverse effects
chemical_list: D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; C542392:vaginolysin, Gardnerella vaginalis