[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Polo-Megías, Daniel; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain. Electronic address: danielpm@ugr.es
Cano-Muñoz, Mario; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain
Trolese, Philipp; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain, Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131, Padova, Italy
Lestani, Sara; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain
La Rocchia, Ilaria; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain
Pierangelini, Andrea; Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131, Padova, Italy
Fongaro, Benedetta; Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131, Padova, Italy
de Laureto, Patrizia Polverino; Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 30131, Padova, Italy
Morales-Yánez, Francisco J; Nano-Antibodies to Explore Protein Structure and Functions (NEPTUNS), Centre for Protein Engineering, InBios, Department of Life Sciences, University of Liège, Liège, Belgium, AlpaNano, Centre for Protein Engineering, InBios, Department of Life Sciences, University of Liège, Liège, Belgium
Vaneyck, Jonathan ; Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
Vanderplasschen, Alain ; Université de Liège - ULiège > Département des maladies infectieuses et parasitaires (DMI) > Immunologie vétérinaire
Decoville, Thomas; Laboratoire d'ImmunoRhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR_S 1109, Institut Thématique Interdisciplinaire (ITI) de Médecine de Précision de Strasbourg, Transplantex NG, Faculté de Médecine, Fédération Hospitalo-Universitaire OMICARE, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, F-67000, Strasbourg, France
Laumond, Géraldine; Laboratoire d'ImmunoRhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR_S 1109, Institut Thématique Interdisciplinaire (ITI) de Médecine de Précision de Strasbourg, Transplantex NG, Faculté de Médecine, Fédération Hospitalo-Universitaire OMICARE, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, F-67000, Strasbourg, France
Salinas-Garcia, M Carmen; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain
Cámara-Artigas, Ana; Department of Chemistry and Physics, University of Almería, Agrifood Campus of International Excellence (ceiA3), Research Center for Mediterranean Intensive Agrosystems and Agri-Food Biotechnology (CIAIMBITAL), Carretera de Sacramento s/n, Almería, 04120, Spain
Gavira, José A; Laboratorio de Estudios Cristalográficos, IACT-CSIC, Armilla, 18100, Granada, Spain
Moog, Christiane; Laboratoire d'ImmunoRhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR_S 1109, Institut Thématique Interdisciplinaire (ITI) de Médecine de Précision de Strasbourg, Transplantex NG, Faculté de Médecine, Fédération Hospitalo-Universitaire OMICARE, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, F-67000, Strasbourg, France, Vaccine Research Institute (VRI), F-94000 Créteil, France
Conejero-Lara, Francisco; Departamento de Química Física, Instituto de Biotecnología y Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain. Electronic address: conejero@ugr.es
H2020 - 681032 - EHVA - European HIV Vaccine Alliance (EHVA): a EU platform for the discovery and evaluation of novel prophylactic and therapeutic vaccine candidates
Funders :
AEI - Agencia Estatal de Investigación Regional Government of Andalusia ESRF - European Synchrotron Radiation Facility ERDF - European Regional Development Fund EU - European Union ULiège - Université de Liège UGR - University of Granada CIP - Canadian Institute of Planners
Funding text :
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 h\u00E9patites 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 \u201CA way to make Europe\u201D and \u201CInvesting in your future.\u201D We are also grateful to the Andalusian Regional Government for the predoctoral fellowship awarded to Daniel Polo-Meg\u00EDas. Mario Cano-Mu\u00F1oz was supported by a Postdoctoral Research Program from the Spanish Research Agency: Juan de la Cierva ( JDC2022-049681-I ). Francisco Morales-Y\u00E1\u00F1ez was supported by a COS-R funding from the University of Li\u00E8ge . 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\u00AE 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 .
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