A glycosylation-associated hemagglutinin substitution improves the breadth of an influenza B mRNA vaccine

Introduction Influenza B virus is an important cause of seasonal influenza, with its contribution varying across regions and seasons. Historically, influenza B viruses have been classified into two major lineages: B/Victoria (BV) and B/Yamagata. No naturally circulating B/Yamagata-lineage viruses have been confirmed after March 2020, making protection against BV the current priority of influenza B vaccination. In our previous study, we demonstrated that the T214A mutation significantly alters the cross-neutralization capacity of this antigen, potentially through altered glycosylation. Notably, the WHO-recommended vaccine strain for the season, B/Austria/1359417/2021 (BV21), lacked the relevant glycosylation position. Methods We used an mRNA platform to compare the cross-subclade protection provided by B/Wyoming/06/2014 (BW06) and BV21 within the B/Victoria lineage. We also introduced the D212N substitution into BV21 as a minimal antigenic modification. Results The D212N substitution restored the glycosylation motif in BV21 and enhanced cross-neutralizing responses against the tested heterologous strains. Mice immunized with a high dose of the D212N-modified vaccine showed 100% survival following challenge with the tested viruses from different subclades. Discussion Our findings provide valuable insights for the development of broadly protective influenza B vaccines.