Efforts to develop broadly protective or universal influenza vaccines have largely emphasized the hemagglutinin (HA) surface glycoprotein. The authors highlight neuraminidase (NA) as an additional antigen capable of eliciting cross-reactive, protective humoral responses. Instead of focusing on HA alone, the work tests whether altering NA antigenicity by adding glycans can bias immune responses toward conserved NA regions that might provide broader protection across strains and subtypes.
The investigators applied an iterative hyperglycosylation strategy to engineer an NA immunogen intended to shield variable surface regions and expose or favor responses to conserved antigenic sites. The engineered NA retained measurable enzymatic activity, indicating preserved functional conformation, and also remained reactive with a conformation-specific antibody known to recognize the conserved catalytic site of NA. These biochemical validations support that glycan additions did not grossly disrupt NA folding or the integrity of at least one conserved functional epitope.
When tested in mice, the hyperglycosylated NA immunogen elicited serum antibody responses of comparable overall magnitude to those elicited by a wild-type NA immunogen. Importantly, the quality of the humoral response differed. Sera from animals immunized with the hyperglycosylated construct showed increased breadth: they recognized N2 NAs from H2N2 and H3N2 viruses spanning nearly 65 years of antigenic drift and also bound a heterosubtypic N9 NA. Thus, glycan shielding altered the antigenic targets of the antibody repertoire in a manner that broadened cross-reactivity across diverse NA variants.
Single B cell analyses of immunized animals identified a monoclonal antibody (mAb) that competed with a component of the serum response elicited by the hyperglycosylated immunogen. Structural characterization localized this mAb to a previously undefined, conserved epitope at the NA tetramer interface. This epitope differs from the canonical catalytic site targeted by many NA-directed antibodies and represents a conserved structural region across multiple NA variants. Identification of an antibody targeting this tetramer-interface site demonstrates that the hyperglycosylation strategy can enrich for responses against novel, conserved NA surfaces.
To evaluate functional relevance, the authors performed passive-transfer experiments. Transfer of the interface-directed monoclonal antibody into mice provided partial protection against a lethal heterologous influenza challenge. The level of protection was partial, indicating that while this single antibody and its epitope can contribute to defense against heterologous virus, additional responses or antibodies likely participate in full protection.
Collectively, the data indicate that targeted glycan shielding can be used as a rational immunogen-design approach to reshape and enrich humoral responses toward a conserved antigenic region on NA. The hyperglycosylated NA immunogen retained enzymatic function and recognition by a conformation-specific catalytic-site antibody while driving broader serologic recognition across decades of N2 antigenic drift and into a heterosubtypic N9. Isolation and structural mapping of an interface-directed mAb, and demonstration of partial protection after passive transfer, provide mechanistic and functional evidence that this design strategy can elicit antibodies against previously underappreciated conserved NA sites. The authors propose that the immunogen and hyperglycosylation approach could serve as a template for next-generation NA-based influenza vaccines that aim to increase breadth of protection.
The preprint declares a competing interest for one author (DL), who reports membership on a scientific advisory board for Metaphore Bio, consultancy relationships with Tendel Therapies and Bio Med X, and unrelated funding from Leyden Labs. The reported funders include the National Institute of Allergy and Infectious Diseases (multiple grants listed) and the National Institute of General Medical Sciences (training grant). The article is a preprint and has not undergone peer review.
The source reports the primary biochemical, immunologic, structural, and passive-transfer findings summarized above. As this content derives from a single preprint, details on experimental methods, sample sizes, statistical analyses, and broader translational testing beyond the described mouse experiments are provided in the full preprint but are not reproduced here. The preprint status indicates the findings have not been peer reviewed.