Rapid synthesis and screening of natively paired antibodies against influenza hemagglutinin stem via oPool+display

Antibody discovery is crucial for developing therapeutics and vaccines as well as understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we presented oPool + display, which combines oligo pool synthesis and mRNA display to construct and characterize the specificity of many natively paired antibodies in parallel. As a proof-of-concept, we applied oPool + display to rapidly screen the binding activity of >300 natively paired influenza hemagglutinin (HA) antibodies against the conserved HA stem domain. Structural analysis of 16.ND.92, one of the identified HA stem antibodies, revealed a unique binding mode distinct from other known broadly neutralizing stem antibodies with the same sequence feature. Yet, despite such differences, 16.ND.92 remained broadly reactive and conferred in vivo protection. Overall, this study not only established an experimental platform to accelerate antibody discovery, but also provides molecular insights into antibody responses to the influenza HA stem, which is a major target for universal influenza vaccine development.