Finally we examined whether sH5 3 would also confer protection in mice. Therefore, 2 groups of 10 mice were vaccinated either once (on day 21) or twice (on day 0 and 21) with 2 mg of sH5 3 adjuvanted with Stimune and challenged three weeks later by intranasal inoculation with ,10 LD 50 of H5N1 A/Viet Nam/ 1194/04. The percentage of mice surviving the infection, median clinical scores and body weights per group observed after the challenge inoculation are shown in Fig. 4 . All mock-vaccinated mice succumbed to infection or had to be euthanized by day 9 p.i.. These mice showed severe clinical signs, including respiratory distress and significant weight loss, which continued until the animals died. A booster vaccination with sH5 3 provided 100% protection against the lethal dose of A/Viet Nam/1194/04; none of the mice showed significant signs of disease and their body weights remained constant. A single vaccination with sH5 3 did not protect mice against disease and concurrent weight loss; yet 40% of the mice survived. These mice started to recover from day 9 p.i. onwards as demonstrated by their regain of body weight. (Fig. 4C ). Fig. 4D shows the pre-challenge anti-sH5 3 antibody levels in individual serum samples,as determined by ELISA. Such antibodies could be detected in most vaccinated animals. However, after a single immunization, these levels remained very low compared to those in animals that received a booster vaccination. These results were essentially confirmed by determining the HI titers against sH5 3 in the same serum samples. With the exception of one animal, the serum of which demonstrated high levels of auto-agglutination, all mice displayed high HI titers when vaccinated twice and low HI titers when vaccinated once. Thus, mice vaccinated twice with sH5 3 were protected against a lethal challenge with HPAI H5N1, while a single vaccination provided partial protection. The differences in protection correlated with the observed serum HI titers.