Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit
The formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1...
| Autores: | , , , , , , , , , , , , , , , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2026 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/420254 |
| Acceso en línea: | http://hdl.handle.net/10261/420254 |
| Access Level: | acceso abierto |
| Palabra clave: | Isothermal titration calorimetry Single-domain antibodies (sdAbs) X-ray crystallography |
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| dc.title.none.fl_str_mv |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| title |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| spellingShingle |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit Polo-Megías, Daniel Isothermal titration calorimetry Single-domain antibodies (sdAbs) X-ray crystallography |
| title_short |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| title_full |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| title_fullStr |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| title_full_unstemmed |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| title_sort |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit |
| dc.creator.none.fl_str_mv |
Polo-Megías, Daniel Cano-Muñoz, Mario Trolese, Philipp Lestani, Sara la Rocchia, Ilaria Pierangelini, Andrea Fongaro, Benedetta de Laureto, P. P. Morales-Yánez, Francisco J. Vaneyck, Jonathan Vanderplasschen, Alain F. C. Decoville, Thomas Laumond, Géraldine Salinas-García, M. C. Cámara-Artigas, Ana Gavira Gallardo, J. A. Moog, Christiane Dumoulin, Mireille Conejero-Lara, Francisco |
| author |
Polo-Megías, Daniel |
| author_facet |
Polo-Megías, Daniel Cano-Muñoz, Mario Trolese, Philipp Lestani, Sara la Rocchia, Ilaria Pierangelini, Andrea Fongaro, Benedetta de Laureto, P. P. Morales-Yánez, Francisco J. Vaneyck, Jonathan Vanderplasschen, Alain F. C. Decoville, Thomas Laumond, Géraldine Salinas-García, M. C. Cámara-Artigas, Ana Gavira Gallardo, J. A. Moog, Christiane Dumoulin, Mireille Conejero-Lara, Francisco |
| author_role |
author |
| author2 |
Cano-Muñoz, Mario Trolese, Philipp Lestani, Sara la Rocchia, Ilaria Pierangelini, Andrea Fongaro, Benedetta de Laureto, P. P. Morales-Yánez, Francisco J. Vaneyck, Jonathan Vanderplasschen, Alain F. C. Decoville, Thomas Laumond, Géraldine Salinas-García, M. C. Cámara-Artigas, Ana Gavira Gallardo, J. A. Moog, Christiane Dumoulin, Mireille Conejero-Lara, Francisco |
| author2_role |
author author author author author author author author author author author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Agencia Estatal de Investigación (España) European Commission Ministerio de Ciencia e Innovación (España) Ministerio de Ciencia, Innovación y Universidades (España) Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72] |
| dc.subject.none.fl_str_mv |
Isothermal titration calorimetry Single-domain antibodies (sdAbs) X-ray crystallography |
| topic |
Isothermal titration calorimetry Single-domain antibodies (sdAbs) X-ray crystallography |
| description |
The formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1 region and block its interaction with HR2, inhibiting viral fusion. Moreover, they are recognized by plasma antibodies from COVID-19 convalescent patients. In this work, we generated camelid heavy-chain-only antibody fragments (VHHs), also named nanobodies (NBs), against a CoVS-HR1 variant mimicking the full HR1 region. A first generation of selected NBs bound HR1 with high affinity and competed with HR2. Notably, this set of NBs exclusively recognized the C-terminal half of HR1, and two of them showed mild neutralizing activity in cell infection assays. Using a truncated CoVS-HR1 variant (N2C), we selected a second generation of NBs targeting specifically the N-terminal half of HR1. However, these NBs did not demonstrate neutralizing activity, possibly due to their low binding affinities. Several NB epitopes were delineated by hydrogen‑deuterium exchange and mass spectrometry analysis, and the crystal structure of a ternary complex between an HR1-mimetic protein and two NBs was determined, confirming competition with HR2. Intriguingly, we found cooperative binding effects between NBs targeting each half of HR1, but these did not result in detectable inhibitory synergy. These findings demonstrate the existence of neutralizing epitopes in the S2 HR1 region and provide a foundation for future development of enhanced neutralizing NBs focused on specific epitopes using HR1-mimetic proteins. |
| publishDate |
2026 |
| dc.date.none.fl_str_mv |
2026 2026 2026 2026 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/article http://purl.org/coar/resource_type/c_6501 Publisher's version info:eu-repo/semantics/publishedVersion |
| format |
article |
| status_str |
publishedVersion |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10261/420254 |
| url |
http://hdl.handle.net/10261/420254 |
| dc.relation.none.fl_str_mv |
#PLACEHOLDER_PARENT_METADATA_VALUE# #PLACEHOLDER_PARENT_METADATA_VALUE# info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-107515RB-C21 info:eu-repo/grantAgreement/EC/H2020/681032 http://dx.doi.org/10.1016/j.ijbiomac.2025.150022 Sí |
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info:eu-repo/semantics/openAccess |
| eu_rights_str_mv |
openAccess |
| dc.publisher.none.fl_str_mv |
Elsevier BV |
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Elsevier BV |
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reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC instname:Consejo Superior de Investigaciones Científicas (CSIC) |
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Consejo Superior de Investigaciones Científicas (CSIC) |
| reponame_str |
DIGITAL.CSIC. Repositorio Institucional del CSIC |
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DIGITAL.CSIC. Repositorio Institucional del CSIC |
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1869419753052831744 |
| spelling |
Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunitPolo-Megías, DanielCano-Muñoz, MarioTrolese, PhilippLestani, Sarala Rocchia, IlariaPierangelini, AndreaFongaro, Benedettade Laureto, P. P.Morales-Yánez, Francisco J.Vaneyck, JonathanVanderplasschen, Alain F. C.Decoville, ThomasLaumond, GéraldineSalinas-García, M. C.Cámara-Artigas, AnaGavira Gallardo, J. A.Moog, ChristianeDumoulin, MireilleConejero-Lara, FranciscoIsothermal titration calorimetrySingle-domain antibodies (sdAbs)X-ray crystallographyThe formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1 region and block its interaction with HR2, inhibiting viral fusion. Moreover, they are recognized by plasma antibodies from COVID-19 convalescent patients. In this work, we generated camelid heavy-chain-only antibody fragments (VHHs), also named nanobodies (NBs), against a CoVS-HR1 variant mimicking the full HR1 region. A first generation of selected NBs bound HR1 with high affinity and competed with HR2. Notably, this set of NBs exclusively recognized the C-terminal half of HR1, and two of them showed mild neutralizing activity in cell infection assays. Using a truncated CoVS-HR1 variant (N2C), we selected a second generation of NBs targeting specifically the N-terminal half of HR1. However, these NBs did not demonstrate neutralizing activity, possibly due to their low binding affinities. Several NB epitopes were delineated by hydrogen‑deuterium exchange and mass spectrometry analysis, and the crystal structure of a ternary complex between an HR1-mimetic protein and two NBs was determined, confirming competition with HR2. Intriguingly, we found cooperative binding effects between NBs targeting each half of HR1, but these did not result in detectable inhibitory synergy. These findings demonstrate the existence of neutralizing epitopes in the S2 HR1 region and provide a foundation for future development of enhanced neutralizing NBs focused on specific epitopes using HR1-mimetic proteins.This research was funded by grant PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033). Additional support was provided by ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales), the Investissements d'Avenir program administered by the ANR (grant ANR-10-LABX-77), and EHVA (Grant No. 681032, Horizon 2020), with co-funding from the ERDF/ESF under the initiatives “A way to make Europe” and “Investing in your future.” We are also grateful to the Andalusian Regional Government for the predoctoral fellowship awarded to Daniel Polo-Megías. Mario Cano-Muñoz was supported by a Postdoctoral Research Program from the Spanish Research Agency: Juan de la Cierva (JDC2022-049681-I). Francisco Morales-Yáñez was supported by a COS-R funding from the University of Liège. Jonathan Vaneyck was supported by CIP funding. Mireille Dumoulin is a research associate from the FRS-FNRS. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain, and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of beamtime and staff assistance at XALOC (ALBA, BAG number 2023087670) and ID30B and ID23-2 (ESRF, BAG number MX2650) beamlines during diffraction data collection. We also acknowledge the Robotein® platform of the BE Instruct-ERIC Centre for providing access to the EasyPick Microlab STARlet Hamilton workstation (https://www.robotein.uliege.be/cms/c_14301428/en/robotein). Funding for open access charge: Universidad de Granada/CBUA.Elsevier BVAgencia Estatal de Investigación (España)European CommissionMinisterio de Ciencia e Innovación (España)Ministerio de Ciencia, Innovación y Universidades (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2026202620262026info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/420254reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-107515RB-C21info:eu-repo/grantAgreement/EC/H2020/681032http://dx.doi.org/10.1016/j.ijbiomac.2025.150022Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4202542026-05-22T06:33:51Z |
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15,812429 |