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Broadly neutralizing antibodies are critical for protection against both drifted and shifted influenza viruses. Here, we reveal that first exposure to the 2009 pandemic H1N1 influenza virus recalls memory B cells that are specific to the conserved receptor-binding site (RBS) or lateral patch epitopes of the hemagglutinin (HA) head domain. Monoclonal antibodies (mAbs) generated against these epitopes are broadly neutralizing against H1N1 viruses spanning 40 years of viral evolution and provide potent protection in vivo. Lateral patch-targeting antibodies demonstrated near universal binding to H1 viruses, and RBS-binding antibodies commonly cross-reacted with H3N2 viruses and influenza B viruses. Lateral patch-targeting mAbs were restricted to expressing the variable heavy-chain gene VH3-23 with or without the variable kappa-chain gene VK1-33 and often had a Y-x-R motif within the heavy-chain complementarity determining region 3 to make key contacts with HA. Moreover, lateral patch antibodies that used both VH3-23 and VK1-33 maintained neutralizing capability with recent pH1N1 strains that acquired mutations near the lateral patch. RBS-binding mAbs used a diverse repertoire but targeted the RBS epitope similarly and made extensive contacts with the major antigenic site Sb. Together, our data indicate that RBS- and lateral patch-targeting clones are abundant within the human memory B cell pool, and universal vaccine strategies should aim to drive antibodies against both conserved head and stalk epitopes.

Summary Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes., Graphical Abstract, Highlights • Antibodies targeting conserved hemagglutinin epitopes are polyreactive • Polyreactive antibodies are preferentially induced by novel influenza viruses • Polyreactivity increases antibody flexibility, affinity, and neutralization potency • Polyreactive naive B cells are selected into the memory B cell pool, Polyreactivity is a common feature of antibodies, but little is known about the selection, function, and role of polyreactive B cells against pathogens. Guthmiller et al. demonstrate polyreactive B cells are selected against broadly neutralizing epitopes of influenza viruses and are linked to increased viral-binding breadth, affinity, and antibody flexibility.

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By comprehensively analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we find mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes are polyreactive. Polyreactive mAbs are preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augments mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

Broadly neutralizing antibodies are critical for protection against drifted and novel influenza viruses. Here, we reveal humans have pre-existing immunity against two broadly neutralizing epitopes of the HA head: the lateral patch and receptor-binding site (RBS). Monoclonal antibodies generated against these epitopes are broadly neutralizing against 40+ years of H1N1 evolution and provide potent protection in vivo. First exposure to the novel 2009 pandemic H1N1 virus recalls memory B cells that target the conserved RBS and lateral patch epitopes. We identified unique repertoire, structural, and binding features of lateral patch and RBS binding antibodies including a lateral patch binding motif shared across subjects. Together, our data indicate that the conserved RBS and lateral patch epitopes are readily targeted by the human memory B cell pool and that universal vaccine strategies should aim to drive antibodies against both conserved head and stalk domain epitopes.

Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however, our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this, we developed the Spec-seq framework, which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here, we present the first application of the Spec-seq framework, which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However, we did find enhanced transcriptional similarity between clonally related B cells, as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine–positive plasmablasts are largely similar, whereas IgA vaccine–negative cells appear to be transcriptionally distinct from conventional, terminally differentiated, antigen-induced peripheral blood plasmablasts.