These results prompted us to determine the minimal sH5 3 dose required to confer protection and to examine whether a single dose could already be sufficient. In the second vaccination-challenge experiment, chickens were thus vaccinated with 10, 2 or 0.4 mg of sH5 3 either once or twice and challenged four weeks later by infection with a lethal dose of A/Viet Nam/1194/04 as described above. The results are shown in Fig. 3 . Again, all mock-vaccinated birds succumbed to the infection within 2 days. Vaccinating twice with a dose of 0.4 mg of sH5 3 was sufficient to protect 90% of the chickens against mortality, while all chickens survived when a dose of 2 or 10 mg was similarly administered (Fig. 3A) . Interestingly, also single vaccination with sH5 3 could induce sufficient immunity to protect chickens against lethal infection (Fig. 3B) ; when a dose of 2 mg was given only one chicken died (90% protection), whereas a dose of 10 mg was protective to all birds. Even after a single dose of 0.4 mg, 60% of the chickens were protected against death.