Immunizations and Influenza Virus Challenge
Mice were immunized subcutaneously (s.c.) at the base of the tail with 2 μg Influenza A H1N1 (A/Puerto Rico/8/1934) Hemagglutinin (HA) (Sino Biological) formulated in the cationic liposomal adjuvant CAF01, containing dimethyldioctadecylammonium (DDA) bromide (250 μg) and trehalose 6,6’-dibehenate (TDB) (50 μg), produced in-house (Statens Serum Institut, Copenhagen, Denmark) as described11. The vaccine was administered in a volume of 200 μl TRIS buffer (isotonic, pH 7.4, with 2% glycerol). Mice were challenged with 30 µL (15 µL/nostril) of the mouse-adapted influenza virus strain A/Puerto Rico/8/1934 (PR8) (150 EID50). The virus was propagated in the allantoic cavity of 10-day-old embryonate hen’s eggs. Allantoic fluid was harvested, clarified, and frozen at −80 ℃ until use.