Female, specified pathogen-free (SPF) 9-week-old BALB/c mice (Charles River Laboratories; 10 animals per group) were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection (0.2 ml) of 2 mg sH5 3 adjuvanted with Stimune. As a challenge control, one group of mice was mock vaccinated. Three weeks after the vaccination, mice were anaesthetized with ketamin/xylazin by intraperitoneal injection and inoculated intranasally with 50 ml of H5N1A/Viet nam/1194/04 containing ,10 median lethal dose (LD 50 ; 3.7 10 log TCID 50 ; provided by Dr. Alan Hay from the WHO Influenza Centre at the National Institute for Medical Research, London). The mice were weighed daily and examined for signs of illness during the next 14 days. Clinical signs were recorded using a scoring system (0 = no clinical signs; 1 = rough coat; 2 = rough coat, less reactive, passive during handling; 3 = rough coat, rolled up, laboured breathing, passive during handling; 4 = rough coat, rolled up, laboured breathing, unresponsive). Animals reaching a score of 4 were euthanized. Surviving animals were bled and sacrificed on day 14 p.i.