ORIGINAL RESEARCH article
Front. Immunol.
Sec. Vaccines and Molecular Therapeutics
Susceptibility of broad reactivity nanobodies to resistance mutations in the S2 domain of SARS-CoV-2 predicted by yeast display deep mutational scanning
Provisionally accepted
- 1National Institute for Biological Standards and Control (NIBSC), Potters Bar, United Kingdom
- 2Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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The rapid evolution of SARS-CoV-2 has led to the erosion of vaccine induced serum neutralization and monoclonal antibody efficacy. As such, interest is inevitably moving towards more conserved regions of the SARS-CoV-2 spike protein like the S2 domain. Resistance mutations continue to be a major obstacle for the development of antivirals and vaccines which target the RBD but what extent these will be a problem for S2 binding antibodies is not known. We have developed a yeast display deep scanning mutagenesis platform which allows an unbiased prospective assessment of millions of single and double mutations for their effects on antibody binding to the S2 domain. We have compared the mutational resistance of a panel of five nanobodies mapped to four distinct non-competing epitopes within the conserved fusion peptide, stem helix and heptad repeat 2 elements of the S2 domain. Yeast display deep mutational scanning predicted reduced binding of C303, G223, G225, and G142 to naturally occurring resistance mutations which were experimentally confirmed on SARS-CoV-2 variants. Our study shows that resistance mutations in conserved elements of the S2 domain may still pose a challenge to the development of monoclonal antibodies and subunit vaccines.
Keywords: deep mutational scanning, Nanobody, pandemicpreparedness, S2, SARS-CoV-2, Yeast display
Received: 16 Oct 2025; Accepted: 10 Dec 2025.
Copyright: © 2025 Hufton, Ball, Ramage and Mate. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Simon Evan Hufton
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