Egyptian rousette bat humoral immunity to H9 influenza hemagglutinin

In mammals, antibodies are central to antiviral defense, but they can also impose selective pressure that drives viral evolution. The interplay between viral antigenic variation and host antibody diversification constitutes a molecular arms race that influences pathogenicity, transmission, and spillover risk. Bats are reservoirs for zoonotic viruses with pandemic potential yet they appear to tolerate infection without overt disease. Although distinctive features of bat innate immunity have been described, the role of adaptive immunity-particularly antibody-mediated responses-remains largely undefined. Moreover, how antibody evolutionary pressure operates in bats is unknown, in part because tools to interrogate bat B cell responses at the monoclonal level are limited. Here, we developed a yeast surface display library of bat antibodies derived from splenic RNA of wild-caught Egyptian rousette bats to interrogate humoral responses to the bat-derived H9 influenza hemagglutinin. We isolated monoclonal antibodies recognizing the hemagglutinin (HA) antigen and defined their gene usage, somatic hypermutation frequency, binding affinities, and breadth. We then used cryo-EM to structurally characterize three bat antibodies in complex with HA engaging distinct antigenic sites. Together, these data enable direct comparison with human anti-influenza antibodies highlighting similarities in humoral immunity across mammals and provides a tool to examine bat antibody responses to other potential zoonotic viruses.