Fig 1.
Serum antibodies confer partial protection against H5N1.
(A) Mice (n = 8/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge. (B) Recipient mice (n = 11-12/group) received 400μl immune sera of 1-time or 2-times TVV+MM immunized donors or naïve serum intraperitoneally one day before challenge. Mice were challenged with 12.5xLD50 of wild type H5N1 A/Hong Kong/156/97 and monitored for 21 days for survival and weight-loss. Graphs A and B represent the Kaplan-Meier survival curves and graphs C and D represent mean bodyweight change with 95% confidence interval. ST = serum transfer.
Fig 2.
Cross-reactive HA and NA-specific ADCC responses are induced by TVV+MM.
Mice (n = 8/group) were immunized once or twice with TVV or TVV+MM. Three weeks later, serum samples were obtained and tested for H5N1 A/Hong Kong/156/97 cross-reactive HAI responses (A), neutralizing antibody responses (B), HA-specific antibodies (C) and NA-specific antibodies (D) or ADCC responses against H5 expressing cells (E) or N1 expressing cells (F). Black bars indicate medians of log-2 transformed HAI and neutralizing titers (NT) or log-10 transformed ELISA titers (EU). Error bars in ADCC assays indicate the standard error of the duplicate means. Control in HAI assay = H5/HK specific sheep serum.
Fig 3.
H5 and N1 cross-reactive T cells are induced by TVV+MM.
Mice (n = 8/group) were immunized once or twice with TVV or TVV+MM. Three weeks later, spleens were harvested. The number of IFN-γ producing T cells was determined by ex vivo stimulation of splenocytes with peptide pools consisting of 15-mer peptides that cover the total (A) HA or (B) NA sequence of H5/HK with an 11-mer overlap. Black bars indicate medians of IFN-γ+ T cells per 106 splenocytes. SFU = Spot forming units.
Fig 4.
CD4+ and CD8+ T cells both contribute to H5N1 protection.
Mice (n = 8-10/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge and (A) CD8+ or the combination of CD4+ and CD8+ T cells were depleted or (B) CD4+ T cells only were depleted with antibodies injected 4 days and 1 day before challenge. Mice were challenged with 12.5xLD50 of wild type H5N1 A/Hong Kong/156/97 and monitored for 21 days for (A and B) survival and (C and D) bodyweight-loss. Graphs A and B represent the Kaplan-Meier survival curves and graphs C and D represent mean bodyweight change with 95% confidence interval.