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

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 <i>in vitro</i> 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 ( <i>P</i> < 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, <i>in vitro</i> platforms in expediting therapeutic antibody development under conditions of high biosafety requirements.