We analyzed whether vaccination with sH5 3 decreased or prevented chickens from shedding challenge virus. For practical reasons, virus shedding was analyzed by a quantitative RT PCR rather than by measuring infectious virus titers. To this end, trachea and cloaca swabs were taken from the chickens of the vaccine dose titration experiment of Fig. 3 at 2, 4 and 7 days after the challenge inoculation. The tracheal and the cloacal swab sampled from each chicken at each particular day were pooled and the presence of viral RNA in the pooled swabs was analyzed using a quantitative RT PCR assay detecting the M1 gene. The results are shown in Table 1 . Of the chickens that received a booster vaccination, only 2 birds, both of which had received the lowest amount of antigen, tested positive. Notably, these were the two animals that developed the lowest sH5 3 -specific antibody titers (Fig. 3C) . One of these animals did not survive the challenge. Virus shedding could not be detected in any of the other birds, although 3 swabs gave inconclusive results. Of the chickens vaccinated only once, all animals that died tested positive. None of the birds vaccinated with 10 mg sH5 3 tested positive. Of the chickens vaccinated with a lower dose and surviving, two tested positive, but only at day 2 p.i. In conclusion, the vaccinated birds that could control the lethal HPAI H5N1 challenge infection exhibited minimum or no virus shedding.