Influenza virus undergoes frequent changes in its main surface glycoprotein, hemagglutinin (HA), evading pre-existing immunity acquired by a previous infection or vaccination. To overcome this challenge, we developed a novel vaccination strategy based on recombinant mosaic HAs (mHAs) that are stabilized without the use of an exogenous trimerization domain (foldon). These stabilized mHAs maintained high trimeric HA content and demonstrated enhanced stability at elevated temperatures and low pH. In mice, sequential immunization with stabilized mHAs redirected the antibody response towards conserved HA regions, resulting in broad protection against diverse influenza virus strains. Compared with foldon-containing constructs, stabilized mHAs improved epitope exposure while avoiding unwanted anti-foldon immune responses. We further compared the immune responses elicited by stabilized mHAs with those induced by conventional seasonal influenza vaccines. Notably, stabilized mHA vaccines conferred protection in the absence of hemagglutination inhibition antibodies, unlike seasonal influenza vaccines. This protection was mediated by a combination of serum neutralization and Fc effector functions predominantly targeting the HA stalk domain. Stabilized mHAs also provided broad cross-protection against homologous (pH1N1), heterologous (SwH1N2), and cross-subtype (H5N1) virus challenges in the mouse model. Overall, stabilized mHAs represent a promising alternative for the development of next-generation universal influenza virus vaccines.
