Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape

Fan Bu; Divyasha Saxena; Hailey Turner-Hubbard; Amy Delaney; Lalit Batra; Charlie Fricke; Skyler Moye; Abhishek Verma; Stanley Perlman; Janarjan Bhandari; Bin Liu; Gang Ye; Jian Zheng; Fang Li

doi:10.1371/journal.ppat.1014223

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Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape

Journal article

Open access

Peer reviewed

Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape

Fan Bu, Divyasha Saxena, Hailey Turner-Hubbard, Amy Delaney, Lalit Batra, Charlie Fricke, Skyler Moye, Abhishek Verma, Stanley Perlman, Janarjan Bhandari, …

PLoS pathogens, Vol.22(5), e1014223

05/18/2026

DOI: 10.1371/journal.ppat.1014223

PMCID: PMC13193604

PMID: 42149970

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Abstract

SARS-CoV-2 continues to accumulate spike mutations that erode the efficacy of antibody therapeutics. The Q493E mutation in the spike RBD, present in recent Omicron subvariants, enables escape from many antibodies and nanobodies, including our Nanosota-9A nanobody, which neutralizes Omicron JN.1 (Q493) but not KP.3 (E493). To address this, we applied a structure-guided in vitro evolution strategy to engineer Nanosota-9A, generating Nanosota-9B, which binds the KP.3 RBD with high affinity but shows reduced binding to JN.1 RBD. To regain breadth, we engineered a bispecific nanobody combining Nanosota-9A and -9B, which effectively neutralizes both JN.1 and KP.3 in infection assays. Our results provide proof of concept for an "update and reuse" strategy: applying structure-guided engineering to update and reuse validated nanobodies to overcome variant escape. This strategy offers a practical path to maintain therapeutic coverage as the virus evolves, supporting more efficient use of research resources and faster responses to emerging variants.

Mutation

Antibodies, Neutralizing - immunology

Antibodies, Viral - genetics

Antibodies, Viral - immunology

COVID-19 - immunology

COVID-19 - virology

Directed Molecular Evolution

Humans

SARS-CoV-2 - genetics

SARS-CoV-2 - immunology

Single-Domain Antibodies - chemistry

Single-Domain Antibodies - genetics

Single-Domain Antibodies - immunology

Spike Glycoprotein, Coronavirus - chemistry

Spike Glycoprotein, Coronavirus - genetics

Spike Glycoprotein, Coronavirus - immunology

Details

Title: Subtitle: Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape
Creators: Fan Bu - University of Minnesota Medical Center
Divyasha Saxena - University of Louisville
Hailey Turner-Hubbard - University of Minnesota
Amy Delaney - University of Minnesota
Lalit Batra - University of Louisville
Charlie Fricke - University of Iowa
Skyler Moye - University of Iowa
Abhishek Verma - University of Iowa
Stanley Perlman - University of Iowa
Janarjan Bhandari - University of Minnesota
Bin Liu - Hormel (United States)
Gang Ye - University of Minnesota Medical Center
Jian Zheng - University of Louisville
Fang Li - University of Minnesota
Resource Type: Journal article
Publication Details: PLoS pathogens, Vol.22(5), e1014223
DOI: 10.1371/journal.ppat.1014223
PMID: 42149970
PMCID: PMC13193604
NLM abbreviation: PLoS Pathog
ISSN: 1553-7374
eISSN: 1553-7374
Publisher: PLoS
Grant note: National Institute of Allergy and Infectious Diseases: R01AI157975
This study was supported by funding from NIH grants R01AI157975 (to F.L.) and U19AI171954 (to F.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Language: English
Date published: 05/18/2026
Academic Unit: Microbiology and Immunology; Stead Family Department of Pediatrics; Iowa Neuroscience Institute; Infectious Disease (Pediatrics)
Record Identifier: 9985164716202771

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