Vaccine and antibody discovery targeting influenza virus neuraminidase

Influenza virus neuraminidase (NA) is a critical antiviral drug target and has recently gained attention as a key component of antibody-mediated protective immunity. We have explored the structural and antigenic landscape of NA across various non-bat subtypes, revealing diverse tetrameric conformations, including an "open" state that may account for variations in NA stability. Using homology-directed protein design, we elucidate the structural principles governing these conformations and stabilize multiple recombinant NAs in a native "closed" state. This stabilization yields two near-atomic resolution structures of NA via cryo-EM, enhancing thermal stability and improving affinity to protective human antibodies. Furthermore, stabilized NAs can be integrated into viruses without compromising fitness. Our findings offer insights into NA structure, stability, and antigenicity, and establish strategies for reinforcing the conformational integrity of recombinant NA. Additionally, by using the recombinant NA, we isolate NA-specific human B cells and identify antibodies that target the underside of the NA globular head domain. These antibodies inhibit viral propagation across a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and provide both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of these antibodies in complex with NA reveal non-overlapping epitopes on the underside of the NA head, which are highly conserved yet typically inaccessible unless the NA head tilts or dissociates. These findings extend our understanding of hidden conserved sites of vulnerability on NA, guiding the development of effective countermeasures against continuously evolving influenza viruses. Together, our research offers a comprehensive view of NA's structural and antigenic properties, paving the way for improved antiviral strategies.