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Publications

Journal Article Ppublish

PubMed: 40329514 DOI: 10.1080/19420862.2025.2499595

Integrating immune library probing with structure-based computational design to develop potent neutralizing nanobodies against emerging SARS-CoV-2 variants.

MAbs 17(1):2499595

Authors: Lidia Cerdán, Katixa Silva, Daniel Rodríguez-Martín, Patricia Pérez, María A Noriega, Ana Esteban Martín, Alfonso Gutiérrez-Adán, Yago Margolles, Juan A Corbera, Miguel A Martín-Acebes, Juan García-Arriaza, Juan Fernández-Recio, Luis A Fernández, José M Casasnovas

Affiliations

  • Department of Microbial Biotechnology, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
  • Department of Macromolecular Structures, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
  • Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
  • Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain.
  • Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas (INIA-CSIC), Madrid, Spain.
  • Department of Animal Reproduction, INIA-CSIC, Madrid, Spain.
  • Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS), Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria (ULPGC), Campus Universitario de Arucas, Gran Canaria, Spain.
  • Instituto de Ciencias de la Vid y del Vino (ICVV), CSIC - Universidad de La Rioja - Gobierno de La Rioja, Logroño, Spain.

Abstract

To generate antibodies (Abs) against SARS-CoV-2 emerging variants, we integrated multiple tools and engineered molecules with excellent neutralizing breadth and potency. Initially, the screening of an immune library identified a nanobody (Nb), termed Nb4, specific to the receptor-binding domain (RBD) of the Omicron BA.1 variant. A Nb4-derived heavy chain antibody (hcAb4) recognized the spike (S) of the Wuhan, Beta, Delta, Omicron BA.1, and BA.5 SARS-CoV-2 variants. A high-resolution crystal structure of the Nb4 variable (VHH) domain in complex with the SARS-CoV-2 RBD (Wuhan) defined the Nb4 binding mode and interface. The Nb4 VHH domain grasped the RBD and covered most of its outer face, including the core and the receptor-binding motif (RBM), which was consistent with hcAb4 blocking RBD binding to the SARS-CoV-2 receptor. In mouse models, a humanized hcAb4 showed therapeutic potential and prevented the replication of SARS-CoV-2 BA.1 virus in the lungs of the animals. , hcAb4 neutralized Wuhan, Beta, Delta, Omicron BA.1, and BA.5 viral variants, as well as the BQ.1.1 subvariant, but showed poor neutralization against the Omicron XBB.1.5. Structure-based computation of the RBD-Nb4 interface identified three Nb4 residues with a reduced contribution to the interaction with the XBB.1.5 RBD. Site-saturation mutagenesis of these residues resulted in two hcAb4 mutants with enhanced XBB.1.5 S binding and virus neutralization, further improved by mutant Nb4 trimers. This research highlights an approach that combines library screening, Nb engineering, and structure-based computational predictions for the generation of SARS-CoV-2 Omicron-specific Abs and their adaptation to emerging variants.

MeSH

Sars-Cov-2, Viruses, Antibodies, Monoclonal, Crystallography, Severe Acute Respiratory Syndrome-Related Coronavirus