Fig. 1: Outline of the study design.

a Adjuvants and vaccine preparation outline. b A phylogenetic tree of N1, based on the nucleotide sequence alignment of the coding region of NA. Strains used in this study are highlighted in green (vaccine strain A/Michigan /45/2015 H1N1), red (used for challenge and serology analysis), and blue (used for serology analysis). Representative strains from major N1 clades (gray) were included for reference to reflect the evolution of the NA. The scale bar shows time in years. c BALB/c mice were intranasally vaccinated twice with rNA-N1-MPP adjuvanted with BDX100 or BDX301 adjuvants in a volume of 10 µl (5 µl/nare). Ovalbumin (OVA) was used as an irrelevant control antigen for the homologous viral challenge study. On day 10 after the boost, nasal washes, BALFs, NALTs, lungs, and spleens were collected for the immunological read-outs. 30 days after the second immunization, mice were challenged with 5 × LD₅₀ of the homologous influenza virus strain A/Singapore/GP1908/2015 H1N1 or heterologous influenza virus strains A/bald eagle/Florida/ W22-134-OP/2022 (H5N1 reassortant with A/Puerto Rico/8/1934 vaccine backbone) and A/New Caledonia/20/1999 (H1N1) for followed by body weight and survival monitoring for two weeks after the challenge. Separate cohorts of mice were infected with 0.5 × LD₅₀ of A/Singapore/GP1908/2015 H1N1 or 0.1 × LD₅₀ of A/bald eagle/Florida/W22-134-OP/2022 (H5N1) or 0.1 × LD₅₀ A/New Caledonia/20/1999 (H1N1) for viral replication analysis in lungs and nasal turbinates 5 days after the challenge. d T-cellular and innate immune response analysis outline. C57B/6 mice were used to assess the T-cellular immune response in lungs and spleens to the rNA-N1-MPP vaccine adjuvanted with BDX100 or BDX301 10 days after the booster immunization and 5 days after the heterologous challenge with 0.1 × LD₅₀ of A/bald eagle/Florida/W22-134-OP/2022 (H5N1) influenza virus strain.