Structure-based design of neuraminidase immunogens for next-generation influenza vaccines

Project Summary Influenza remains a global health challenge. There is a pressing need for next-generation vaccines that can protect against a broad range of influenza subtypes, particularly those of pandemic potential (e.g., H5, H7). Hemagglutinin (HA) and neuraminidase (NA) are two surface glycoproteins, which facilitate virion binding and egress, respectively. While contemporary influenza vaccines are not standardized for NA, recent studies have underscored the importance of NA-directed antibodies in influenza protection. However, relative to HA, the immunodominance of NA and its evolutionary response to immune pressure remain largely uncharacterized. Furthermore, whether an NA-based immunogen(s) can focus the humoral response to conserved sites on NA has yet to be determined. The NA catalytic site (CS) cleaves sialic acid to enable viral egress and is conserved amongst NA subtypes. Here, I hypothesize that structure-guided immunogen design can focus the immune response to the conserved NA CS and augment anti-NA antibody breadth. To elicit a focused, CS-directed immune response, I will pursue two approaches. 1) l will engineer glycans on the NA periphery to mask variable epitopes, leaving the conserved CS exposed. 2) I will graft the conformationally complex CS onto antigenically distinct scaffolds to enrich the CS epitope relative to other epitopes. To elicit broader anti-NA immunity and augment CS-directed B cell activation, I will increase NA valency using protein linkers and nanoparticle platforms. I will then test the effects of these NA immunogens in vivo and characterize the resulting serum, germinal center, and memory B cell repertoires. I further hypothesize that focused anti-NA immunity will complement focused anti-HA immunity to restrict influenza escape pathways. After passaging an influenza strain with anti-HA antibodies, anti-NA antibodies, or both, I will characterize viral escape mutants and compare growth kinetics relative to the wildtype strain. The data generated from this proposal will elucidate the effectiveness of the proposed immune-focusing strategies as a general approach to vaccine design and particularly inform the optimization of NA immunogens for next-generation influenza vaccines. These results will also advance our understanding of NA antigenicity and evolvability.