A rationally designed neuraminidase immunogen elicits humoral responses to a conserved viral site

Efforts to develop a universal influenza vaccine have primarily focused on the surface-exposed viral hemagglutinin (HA), but neuraminidase (NA) is an additional target for cross-reactive, protective responses. Here, we used hyperglycosylation as an immunogen design approach to reshape anti-NA humoral immunity toward conserved antigenic regions. Iterative design produced a hyperglycosylated NA immunogen that retained enzymatic activity and reactivity to a conformation-specific antibody recognizing the conserved catalytic site. In mice, the hyperglycosylated immunogen elicited serum antibody responses of comparable magnitude to those elicited by the wild-type NA immunogen. However, serum antibodies elicited by the hyperglycosylated immunogen had increased breadth, recognizing N2 NAs from H2N2 and H3N2 viruses spanning nearly 65 years of antigenic drift, as well as a heterosubtypic N9 NA. Single B cell analyses identified a monoclonal antibody that competed with a component of the serum antibody response elicited by the hyperglycosylated immunogen, and structural characterization showed that it recognizes a previously undefined, conserved epitope at the NA tetramer interface. Passive transfer of this interface-directed antibody partially protected mice against lethal heterologous influenza challenge. Collectively, these data show that glycan shielding can reshape and enrich humoral responses toward a conserved antigenic region on NA. The immunogen and hyperglycosylation design strategy described here provide a template for developing next- generation NA-based influenza vaccines. ONE SENTENCE SUMMARYA hyperglycosylated influenza neuraminidase immunogen reshapes humoral immunity and elicits antibodies targeting a conserved tetramer-interface epitope.