Isolation of neutralizing antibodies against SARS-CoV-2 through an epitope-guided negative screening by phage display

Abstract

Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness, capable of directly inhibiting viral entry and limiting disease progression. However, traditional antibody discovery strategies—such as animal immunization or B cell isolation from infected individuals—are often hindered by biosafety concerns, lengthy development timelines, and limited adaptability during outbreaks. In the present study, we aimed to establish a robust and rapid in vitro platform for the efficient isolation of neutralizing antibodies targeting conserved viral epitopes. We developed an epitope-guided negative screening strategy that integrates phage display technology with rational antigen mutagenesis to exclude antibodies against variable regions while enriching for those that recognize functionally constrained epitopes. When applied to the receptor-binding domain of severe acute respiratory syndrome coronavirus 2, this method enabled the identification of six neutralizing antibodies (one IgG and five nanobodies) exhibiting broad-spectrum neutralizing activity across multiple viral variants. Notably, antibodies recognizing distinct epitopes demonstrated significant synergistic neutralization when used in combination (P < 0.05). This screening approach facilitates the rapid discovery of potent and mutation-resistant antibodies and holds promise for application to other emerging pathogens. Our findings underscore the potential of epitope-guided, in vitro platforms in expediting therapeutic antibody development under conditions of high biosafety requirements.

Keywords

epitope-guided / neutralizing antibody / SARS-CoV-2 / phage display

Funding

The current study was supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 22KJB310001 to W.G.), the Special Project of the Jiangsu Provincial Department of Science and Technology (Grant No. BE2023603 to W.G.), and the Nanjing Medical University Science and Technology Development Foundation (Grant No. NMUB20210006 to W.G.).

Acknowledgements

The authors acknowledge technical support from the Key Laboratory of Antibody Technology, National Health Commission of China, Nanjing Medical University. We are grateful to Professor Zhibin Hu and Zhenqing Feng (Nanjing Medical University) for their expert guidance in experimental design and data interpretation.

Additional information

The online version contains supplementary material available at http://www.jbr-pub.org.cn/article/doi/10.7555/JBR.39.20250259?pageType=en.

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