Fig. 1: Long-term maintenance of neutralization titers and antigen-specific IgG with a rapid memory recall response on challenge and low virus titers in vaccinated macaques after the virus infection.

a Cynomolgus macaques were subcutaneously vaccinated twice with inactivated whole particles of a low pathogenic avian influenza virus A/duck/Hokkaido/Vac-3/2007 (H5N1) (Vac-3) (H5 clade classical/outlier) with a two-week interval. Plasma was collected on week 0 (before vaccination), 2, 4, 8, and 32 weeks, and 1, 2, 3, 4, and 5 years after the prime-boost vaccination to measure Vac-3-specific IgG and neutralization titers. Five years later, macaques were challenged with an H5N1 highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) (H5 clade 1). Autopsy was performed 7 days after challenge. b–d Virus titers after challenge infection. Nasal swab (b), tracheal swab (c), and bronchial brush (d) samples were collected on the indicated days (Supplementary Table 2). Left: each symbol shows the average titer calculated from titers in vaccinated macaques (red: n = 6 for those challenged 5 years after the prime-boost vaccination and blue: n = 3 for those challenged 5 weeks after the first and 3 weeks after the second vaccinations) and unvaccinated macaques (black: n = 3). Right: area under the virus titer and time curve (AUC). e Vac-3 specific IgG levels before and after challenge indicated as 50% max dilution. f Neutralization titers against Vac-3 before and after challenge infection. Each symbol indicates each macaque, with identification numbers in the graph. Neutralization titers higher than 9 and lower than 2 were calculated as 9 and 1, respectively. Statistical analysis was performed with the Mann-Whitney U test in b–d and the Wilcoxon test in e and f: *p < 0.05, **p < 0.01.