Four ferrets per group were immunized with 60 μg A/Astrakhan/3212/2020 HA mRNA-LNP or Luciferase mRNA-LNP (control mRNA-LNP), followed by a 60 μg boost 28 days later. Ferrets were challenged with A/Bald eagle/North Carolina/ W22–140/2022 28 days after the second vaccination. Ferret serum samples were collected before vaccination and 28 days after each vaccination. (A-B) A/Astrakhan/3212/2020 and (C-D) A/pheasant/New York/22–009066-001/2022 serum IgG binding and neutralizing titers were quantified. Reciprocal dilutions of serum required to inhibit 50% virus infection are shown in panels (B) and (D). (E-F) Vaccinated ferrets were monitored for 14 days after i.n. infection with A/Bald eagle/North Carolina/ W22–140/2022. (E) Survival, (F) body weight, (G) virus titers in nasal wash samples, and (H) clinical scores are reported after infection. Data in (A-D) are shown as geometric means ± 95% confidence intervals and values were log-transformed prior to statistical analysis. Data in (F-G) are shown as means ± SEMs. Data in (A-D, F-G) were analyzed by mixed-model ANOVA with Greenhouse-Geisser correction and Sidak’s multiple comparisons test to compare differences with luciferase (control) mRNA immunization. For animals that died, their weight and viral titers on the day prior to death were carried forward for statistical analyses. Data in (E) were analyzed using a log rank test. *P<0.05.