Mice were vaccinated by TA route with 1 or 10 μg of S1 antigen with or without SF‐10 twice or thrice every 2 weeks. TA vaccination was performed by administering 30 μL of S1‐SF‐10 or saline containing S1 antigen into the nasal cavity of mice using a pipette; the dosage is the amount sufficient covering a mucous membrane from the nasal cavity to the lower respiratory tract (Figure S1 ).
As a positive control group, mice were immunized intramuscularly (into thigh muscles) with 10 μg S1 in 50 μL of saline or 10 μg S1 mixed with an adjuvant AddaS03™ (InvivoGen, San Diego, CA) using a 1 mL plastic syringe. S1‐AddaS03™ solution was prepared according to the instructions provided by the manufacturer. At 2 weeks after the last immunization, serum and bronchoalveolar lavage fluid (BALF) samples were obtained as described in detail previously.15
As a positive control group, mice were immunized intramuscularly (into thigh muscles) with 10 μg S1 in 50 μL of saline or 10 μg S1 mixed with an adjuvant AddaS03™ (InvivoGen, San Diego, CA) using a 1 mL plastic syringe. S1‐AddaS03™ solution was prepared according to the instructions provided by the manufacturer. At 2 weeks after the last immunization, serum and bronchoalveolar lavage fluid (BALF) samples were obtained as described in detail previously.
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