Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin
Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resist...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-07-01
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| Series: | Electronic Journal of Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0717345817300258 |
| _version_ | 1819207476194574336 |
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| author | Zilvinas Dapkunas Aurimas Baranauskas Gitana Mickiene Milda Pleckaityte Gintautas Zvirblis |
| author_facet | Zilvinas Dapkunas Aurimas Baranauskas Gitana Mickiene Milda Pleckaityte Gintautas Zvirblis |
| author_sort | Zilvinas Dapkunas |
| collection | DOAJ |
| description | Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the 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 the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker.
Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and 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 in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv.
Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis. |
| first_indexed | 2024-12-23T05:24:06Z |
| format | Article |
| id | doaj.art-c358b51601e844c0951b198d50eda96f |
| institution | Directory Open Access Journal |
| issn | 0717-3458 |
| language | English |
| last_indexed | 2024-12-23T05:24:06Z |
| publishDate | 2017-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Electronic Journal of Biotechnology |
| spelling | doaj.art-c358b51601e844c0951b198d50eda96f2022-12-21T17:58:38ZengElsevierElectronic Journal of Biotechnology0717-34582017-07-0128C525710.1016/j.ejbt.2017.05.003Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysinZilvinas Dapkunas0Aurimas Baranauskas1Gitana Mickiene2Milda Pleckaityte3Gintautas Zvirblis4Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, Vilnius 10257, LithuaniaProfarma UAB, V. A. Graičiūno 6, Vilnius 02241, LithuaniaInstitute of Biotechnology, Vilnius University, Saulėtekio av. 7, Vilnius 10257, LithuaniaInstitute of Biotechnology, Vilnius University, Saulėtekio av. 7, Vilnius 10257, LithuaniaInstitute of Biotechnology, Vilnius University, Saulėtekio av. 7, Vilnius 10257, LithuaniaBackground: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the 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 the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and 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 in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.http://www.sciencedirect.com/science/article/pii/S0717345817300258Circulation half-lifeELISAGardnerella vaginalisGenetic fusionHemolysisMedical biotechnologyPore-forming toxinRecombinant antibodiesTherapeutic agentVaginal bacteriumVirulence factor |
| spellingShingle | Zilvinas Dapkunas Aurimas Baranauskas Gitana Mickiene Milda Pleckaityte Gintautas Zvirblis Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin Electronic Journal of Biotechnology Circulation half-life ELISA Gardnerella vaginalis Genetic fusion Hemolysis Medical biotechnology Pore-forming toxin Recombinant antibodies Therapeutic agent Vaginal bacterium Virulence factor |
| title | Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin |
| title_full | Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin |
| title_fullStr | Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin |
| title_full_unstemmed | Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin |
| title_short | Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin |
| title_sort | generation of dimeric single chain antibodies neutralizing the cytolytic activity of vaginolysin |
| topic | Circulation half-life ELISA Gardnerella vaginalis Genetic fusion Hemolysis Medical biotechnology Pore-forming toxin Recombinant antibodies Therapeutic agent Vaginal bacterium Virulence factor |
| url | http://www.sciencedirect.com/science/article/pii/S0717345817300258 |
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