There are various mechanisms by which anti-HA antibodies can directly neutralize influenza viruses such as by inhibition of membrane fusion, inhibition of HA0 cleavage and inhibition of egress. breadth of protection over immunization with full length (FL) HA. We show in mice that in a direct comparison to NESP H1 FL HA, using the same immunization regimen, dosing and adjuvant, a group 1 mini-HA has a higher protective efficacy against group 1 influenza computer virus challenges not homologous to the H1 FL HA. Although both antigens induce a similar breadth of HA subtype binding, mini-HA immunization induces significantly more HA stem-specific antibodies 7-Methylguanine correlating with survival. In addition, both mini-HA and H1 FL HA immunization induce influenza neutralizing antibodies while mini-HA induces significantly higher levels of mFcRIII activation, involved in Fc-mediated antibody effector functions. In agreement with previous findings, this confirms that more than one mechanism contributes to protection against influenza. Together our results further warrant the development of a universal influenza vaccine based on the HA stem region. Keywords: universal influenza vaccine, hemagglutinin stem immunogen, influenza, mouse influenza challenge models, antibodies Introduction Influenza is usually a 7-Methylguanine major global health problem causing serious morbidity, mortality, and substantial productivity loss each 7-Methylguanine year. The most cost-effective strategy to prevent influenza is usually by vaccination (1, 2). Although current seasonal vaccines are often effective, a mismatch between circulating computer virus strains and the strains included in the vaccine occasionally reduces vaccine efficacy (3). In addition, seasonal vaccines have limited effectiveness against influenza strains newly introduced in the human population (4). The discovery of influenza HA-specific antibodies in humans which are able to neutralize a broad spectrum of influenza A and B strains (5) has raised hope for a cross-protective influenza vaccine able to elicit these broadly protective antibodies. Such a universal influenza vaccine could mitigate the problems of mismatch between the vaccine and circulating strains, as well as providing protection to novel pandemic influenza strains (6). Interestingly, cross-reactive antibodies can also transiently be induced in mice and humans by one or multiple immunizations with a seasonal vaccine (7C9). Induction of these antibodies in humans correlated with protection of mice against lethal challenge with a genetically distant H5N1 influenza strain after passive antibody transfer (10). However, the majority of antibodies elicited in humans by vaccination or exposure to influenza A bind to the immunodominant and highly variable hemagglutinin (HA) head epitopes, often specific for one computer virus strain (11C13). In contrast, most influenza A cross-reactive broadly neutralizing antibodies (bnAb) target the highly conserved HA stem region, but are present at a relatively low frequency in humans (14C16). Various approaches to induce cross-protective antibodies targeting the less immunogenic stem region were tested such as sequential immunizations with chimeric HA molecules, composed of different HA head regions on the same stem region (17C19), and shielding of the HA head epitopes by hyperglycosylation (20, 21). Another strategy directing the immune response to the more conserved HA stem is usually by removing the immunodominant head region, constructing headless HA stem-based antigens. We recently described a stable trimeric headless group 7-Methylguanine 1 influenza HA, the so-called mini-HA, while others pursued a hemagglutinin-stem nanoparticle based approach; both approaches led to formulations that were immunogenic and cross-protective against different phylogenetically distant influenza computer virus strains in mice, non-human primates (NHP) and ferrets (22C26). However, the epitopes recognized by cross-reactive antibodies are also present in full length HA and cross-protective immune responses can be found in humans and mice after multiple immunizations with a seasonal vaccine (7C9). This has raised the question how the immune response to mini-HA compares to the response to H1 full length (FL) HA as found in seasonal vaccines. In this study we directly compared immunization with H1 FL HA A/Brisbane/59/07 (H1 FL HA) 7-Methylguanine to a group 1 mini-HA which consists of the same backbone (23), using equimolar doses, the same immunization regimen and adjuvant in different influenza challenge mouse models. We show that.