Highly pathogenic avian influenza H5N1 viruses are capable of causing poultry epidemics and human mor-tality. Vaccines that induce protective neutralizing antibodies can prevent outbreaks and decrease the potential for influenza A pandemics. Identifying unique H5N1 virus-specific HLA class II-restricted epitopes is essential for monitoring cellular strain-specific immunity. Our results indicate that 80% of the 30 study participants who were inoculated with an H5N1 vaccine produced neutralizing antibodies. We used intracellular cytokine staining (ICS) to screen and identify six DR1501-restricted H5N1 virus epitopes: H5HA148–162, H5HA155–169, H5HA253–267, H5HA260–274, H5HA267–281 and H5HA309–323. Tetramer staining results confirmed that two immunodominant epitopes were DR1501-restricted: H5HA155–169 and H5HA267–281. Both are located at the HA surface and are highly conserved in currently circulating H5N1 clades. These results suggest that a combination of ICS and tetramer staining can be used as a T-cell epitope-mapping platform, and the identified epitopes may serve as markers for monitoring vaccine efficacy.
