Heterosubtypic protection by post-vaccination serum is not boosted by subsequent immunizations and is lost after the second vaccination.

<p>(<b>A</b>) Serum was obtained from 25 human subjects immediately prior to the first vaccination and 4 weeks after each of three vaccinations (pre-vaccination, 1×, 2×, and 3×, respectively). (<b>B</b>) Human-to-mouse serum transfer was used to assess humoral protection against vaccine homologous H1N1 A/Netherlands/602/2009 and heterosubtypic H5N1 A/Hong Kong/156/97 influenza viruses. To assess the protective activity of the sera, 400 µl of each serum sample was transferred to each of two individual mice which were challenged 24 h later with 25×LD₅₀ of vaccine homologous A/Netherlands/602/2009 (H1N1) or heterosubtypic A/Hong Kong/156/97 (H5N1), respectively. All post-vaccination groups show comparable protection against H1N1 challenge. There is a significant difference in protection against H5N1 among different vaccination groups (exact logistic regression, p = 0.035).