To produce antigen for in vitro assays, the H1-SS antigen (Gen6) was also fused to C-terminal thrombin cleavage site, followed by the trimeric foldon domain of T4 fibritin and a hexahistidine tag sequence. Plasmids were purified with the PowerPrep Kit (OriGene #NP100009) and used to transfect 293Expi cells (Thermo Fisher Scientific, #A14635). FectoPRO DNA transfection reagent (Polyplus, #116-100) was used (0.5 µg of DNA/ml, 0.75 µl of FectoPRO reagent/ml, and 0.45 µl of enhancer/ml). Four days after transfection, supernatant was harvested by centrifugation at 3488g for 15 min at 4°C and filtered through a 0.45-µm vacuum-driven filter unit (Thermo Fisher Scientific, #167-0045).

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus–associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential  for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.