To examine the immunogenicity of sH5 3 and its potential as a vaccine a first experiment in chickens was performed, in which 10 chickens were vaccinated twice (on day 0 and 21) i.m. with 10 mg of Stimune-adjuvanted sH5 3 and challenged 3 weeks later by i.n./i.t. inoculation of a lethal dose of A/Viet Nam/1194/04 virus. Another 10 birds were mock-vaccinated to serve as challenge controls. As shown in Fig. 2 , the boost vaccination with 10 mg sH5 3 conferred complete protection. None of the vaccinated chickens died or showed symptoms indicative of influenza-related disease, whereas all mockvaccinated chickens succumbed within 2 days. Serological analysis showed that none of the mock-vaccinated animals contained antibodies against sH5 3 as determined by a sH5 3 -specific ELISA (Fig. 2B) . In contrast, all immunized animals demonstrated appreciable levels of HA antibodies already after a single immunization and these levels increased further after the boost. These total antibody levels against sH5 3 correlated nicely with the HI titers against H5N1 (Fig. 2C) . All mock-vaccinated chickens had a HI titer below the detection limit. HI antibody titers observed after the sH5 3 boost reached with a maximum of 1024 and a minimum of 64, which was apparently still sufficient to protect the animal against the lethal challenge. Interestingly, 50% of the birds developed HI antibody titers equal to 64 or higher already three weeks after the first vaccination. In addition, HI titers were also determined by using sH5 3 rather than H5N1 virus as the hemagglutinating agent, which gave essentially similar results (compare Fig. 2D and 2C) , demonstrating the reliability of the assay. In summary, the results show that chickens vaccinated twice with sH5 3 are protected against a lethal challenge with H5N1. The HI titers observed suggested that one vaccination might already be sufficient to confer protection against HPAI H5N1.