Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previo...

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Autores principales:	Kohei Yumoto, Tomoaki Arisaka, Kazuma Okada, Kyosuke Aoki, Toyoyuki Ose, Tatsunori Masatani, Makoto Sugiyama, Naoto Ito, Hideo Fukuhara, Katsumi Maenaka
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	rabies virus rabies virus glycoprotein single-chain Fv Fab neutralization antibody biolayer interferometry (BLI) Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bd023514215e4b4cb3ff69781dec2dcf
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spelling	oai:doaj.org-article:bd023514215e4b4cb3ff69781dec2dcf2021-11-25T19:14:28ZCharacterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein10.3390/v131123111999-4915https://doaj.org/article/bd023514215e4b4cb3ff69781dec2dcf2021-11-01T00:00:00Zhttps://www.mdpi.com/1999-4915/13/11/2311https://doaj.org/toc/1999-4915Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15–13 and 12–22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15–13 and 12–22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm–baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15–13 Fab interacts with the RABVG, with a <i>K</i><sub>D</sub> value at the nM level, and that the 12–22 Fab has a weaker binding affinity (<i>K</i><sub>D</sub> ~ μM) with the RABVG compared to the 15–13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15–13 and 12–22 antibodies as another potential biopharmaceutical for targeting rabies. The 15–13 and 12–22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the <i>K</i><sub>D</sub>, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.Kohei YumotoTomoaki ArisakaKazuma OkadaKyosuke AokiToyoyuki OseTatsunori MasataniMakoto SugiyamaNaoto ItoHideo FukuharaKatsumi MaenakaMDPI AGarticlerabies virusrabies virus glycoproteinsingle-chain FvFabneutralization antibodybiolayer interferometry (BLI)MicrobiologyQR1-502ENViruses, Vol 13, Iss 2311, p 2311 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	rabies virus rabies virus glycoprotein single-chain Fv Fab neutralization antibody biolayer interferometry (BLI) Microbiology QR1-502
spellingShingle	rabies virus rabies virus glycoprotein single-chain Fv Fab neutralization antibody biolayer interferometry (BLI) Microbiology QR1-502 Kohei Yumoto Tomoaki Arisaka Kazuma Okada Kyosuke Aoki Toyoyuki Ose Tatsunori Masatani Makoto Sugiyama Naoto Ito Hideo Fukuhara Katsumi Maenaka Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
description	Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15–13 and 12–22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15–13 and 12–22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm–baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15–13 Fab interacts with the RABVG, with a <i>K</i><sub>D</sub> value at the nM level, and that the 12–22 Fab has a weaker binding affinity (<i>K</i><sub>D</sub> ~ μM) with the RABVG compared to the 15–13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15–13 and 12–22 antibodies as another potential biopharmaceutical for targeting rabies. The 15–13 and 12–22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the <i>K</i><sub>D</sub>, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.
format	article
author	Kohei Yumoto Tomoaki Arisaka Kazuma Okada Kyosuke Aoki Toyoyuki Ose Tatsunori Masatani Makoto Sugiyama Naoto Ito Hideo Fukuhara Katsumi Maenaka
author_facet	Kohei Yumoto Tomoaki Arisaka Kazuma Okada Kyosuke Aoki Toyoyuki Ose Tatsunori Masatani Makoto Sugiyama Naoto Ito Hideo Fukuhara Katsumi Maenaka
author_sort	Kohei Yumoto
title	Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
title_short	Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
title_full	Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
title_fullStr	Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
title_full_unstemmed	Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein
title_sort	characterization of single-chain fv fragments of neutralizing antibodies to rabies virus glycoprotein
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/bd023514215e4b4cb3ff69781dec2dcf
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Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein

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