Cargando…
Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines
Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased im...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9611486/ https://www.ncbi.nlm.nih.gov/pubmed/36297421 http://dx.doi.org/10.3390/pharmaceutics14101985 |
_version_ | 1784819538592792576 |
---|---|
author | Manieri, Tania Maria Takata, Daniela Yumi Targino, Roselaine Campos Quintilio, Wagner Batalha-Carvalho, João Victor da Silva, Camila Maria Lucia Moro, Ana Maria |
author_facet | Manieri, Tania Maria Takata, Daniela Yumi Targino, Roselaine Campos Quintilio, Wagner Batalha-Carvalho, João Victor da Silva, Camila Maria Lucia Moro, Ana Maria |
author_sort | Manieri, Tania Maria |
collection | PubMed |
description | Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb. |
format | Online Article Text |
id | pubmed-9611486 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-96114862022-10-28 Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines Manieri, Tania Maria Takata, Daniela Yumi Targino, Roselaine Campos Quintilio, Wagner Batalha-Carvalho, João Victor da Silva, Camila Maria Lucia Moro, Ana Maria Pharmaceutics Article Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb. MDPI 2022-09-20 /pmc/articles/PMC9611486/ /pubmed/36297421 http://dx.doi.org/10.3390/pharmaceutics14101985 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Manieri, Tania Maria Takata, Daniela Yumi Targino, Roselaine Campos Quintilio, Wagner Batalha-Carvalho, João Victor da Silva, Camila Maria Lucia Moro, Ana Maria Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title_full | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title_fullStr | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title_full_unstemmed | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title_short | Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines |
title_sort | characterization of neutralizing human anti-tetanus monoclonal antibodies produced by stable cell lines |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9611486/ https://www.ncbi.nlm.nih.gov/pubmed/36297421 http://dx.doi.org/10.3390/pharmaceutics14101985 |
work_keys_str_mv | AT manieritaniamaria characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT takatadanielayumi characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT targinoroselainecampos characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT quintiliowagner characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT batalhacarvalhojoaovictor characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT dasilvacamilamarialucia characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines AT moroanamaria characterizationofneutralizinghumanantitetanusmonoclonalantibodiesproducedbystablecelllines |