Broadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is anchored to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an under appreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

Artificial mutagenesis and chimeric/mosaic protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used for influenza research and vaccine development require sequence editing and protein expression, limiting their applicability and the progress of related research to specialists. Rapid tools allowing even novice influenza researchers to properly analyze and visualize influenza protein sequences with accurate nomenclature are needed to expand the research field. To address this need, we developed Librator, a system for analyzing and designing protein sequences of influenza virus Hemagglutinin (HA) and Neuraminidase (NA). With the graphical user interface (GUI) and built-in sequence editing functions of Librator, biologists can easily analyze influenza sequences and phylogenies, automatically port sequences to visualize structures, then readily mutate target residues and design sequences for antigen probes and chimeric/mosaic proteins efficiently and accurately. This system provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible, allowing for significant reagent savings. Use of Librator will significantly facilitate influenza research and vaccine antigen design.

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