Mi3 nanoparticles displaying the knob domains of Fiber1 and Fiber2 from fowl adenovirus serotype 4 induce robust humoral and cellular immune responses in chickens.

Hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) has led to substantial economic losses in the poultry industry. Subunit vaccines based on the knob domains of Fiber1 and Fiber2 (F1F2K) are safe but exhibit limited immunogenicity. To address this, we sought to enhance the immune response by presenting F1F2K on self-assembling Mi3 nanoparticles (F1F2K:Mi3) and to compare its efficacy with the F1F2K monomer. F1F2K was conjugated to Mi3 via the SpyTag/SpyCatcher system. Chickens were immunized twice with F1F2K:Mi3 nanoparticles, F1F2K monomer, Mi3, or PBS, and then challenged with a lethal dose of FAdV-4. Humoral and cellular immune responses were evaluated by enzyme-linked immunosorbent assay (ELISA), virus neutralization tests, and cytokine assays. Compared with the F1F2K monomer, F1F2K:Mi3 nanoparticles induced significantly higher antigen-specific IgG titers at 28 days post-vaccination (dpv) and a 2.5-log₂ increase in neutralizing antibody titers. Nanoparticle immunization also elicited stronger T helper 1/T helper 2 (Th1/Th2) cytokine responses (IFN-γ, IL-4, and IL-2). Following challenge, all chickens in both the nanoparticle and monomer groups survived. However, compared with the F1F2K monomer group, the F1F2K:Mi3 group exhibited less weight loss, lower tissue viral loads, reduced viral shedding, and markedly less liver damage, thereby more effectively blocking FAdV-4 pathogenicity in target organs. Our study demonstrates that displaying the F1F2K antigen on Mi3 nanoparticles synergistically enhances both humoral and cellular immune responses against FAdV-4, providing a promising next-generation nanoparticle vaccine strategy for HHS control.