The efficacy of the sH5 3 vaccine was first studied in chickens. Adjuvanted with Stimune, a water-in-oil adjuvant also known as Specol, the sH5 3 protein formulation induced an immunity that was completely protective against a lethal H5N1 challenge after administration of two doses ($2 mg sH5 3 /dose). Importantly, our vaccine preparation also protected chickens after a single immunization. While 100% of the chickens were protected after vaccination with 10 mg sH5 3 , 90% were protected when using 2 mg. Similar HA doses were previously needed to protect chickens with a vaccine preparation consisting of full length HA proteins, which had been purified from insect cell cultures infected with recombinant baculovirus expressing the H5 gene, emulsified in a water-in-oil adjuvant [17] . These results are consistent with the observation that mammalian cell-produced HA trimers elicited similar levels of neutralizing antibodies as trimeric HA produced in insect cells [16] . The efficacy of our sH5 3 vaccine preparation was furthermore demonstrated by the absence of viral RNA in the protected birds. This would imply that a vaccinated flock can pose a barrier against further spread of circulating virus.