A glycoengineered yeast-derived HA1-ferritin nanoparticle vaccine confers protective immunity against H7 avian influenza in chickens

H7 subtype avian influenza virus is an important zoonotic pathogen threatening the poultry industry and public health. Recombinant subunit vaccines provide a safer alternative to conventional inactivated vaccines, while yeast expression systems offer advantages of low cost, scalability, and engineering flexibility. However, their application in avian influenza vaccine development remains underexplored. In this study, we use the methylotrophic yeast Komagataella phaffii as a low-cost, industrial-scale platform to produce hemagglutinin-1 (HA1) subunits with programmable N -/ O -linked glycosylation. The methanol-utilization-slow (Mut S ) phenotype strain secreted 1.21 g/L HA1, and compared with the standard methanol-utilization-plus (Mut + ) phenotype strain, the N -linked and O -linked glycans were significantly reduced [liquid chromatography–mass spectrometry (LC–MS)/MS glycopeptide profile]. Despite hypoglycosylation, the antigen retained native conformation, binding H7-specific mAb Q74 with a K D of 1.34 × 10 –7 M. SpyTag/SpyCatcher-directed conjugation to ferritin drove quantitative self-assembly into 30 nm virus-like nanoparticles. Formulated with CpG-ODN 1826, the HA1-Mut S -ferritin candidate elicited Hemagglutination inhibition (HI) titers of 6.7 log 2 in specific pathogen-free (SPF) leghorns at 28 days post-immunization (dpi), significantly enhancing HA1-specific IgY responses ( p < 0.0001) while sustaining high-level circulating CD4 + T cells. At 28 dpi, chickens were challenged with 10 6 embryo infectious dose (EID) 50 of H7N9 avian influenza virus; the formulation conferred 100% clinical protection and accelerated viral clearance, matching the performance of conventional inactivated vaccines. Yeast-based processes significantly reduce downstream purification costs and eliminate reliance on chicken embryos, providing a scalable and biosafe platform for rapid avian influenza control.