Identifying conserved DR1501-restricted CD4⁺ T-cell epitopes in avian H5N1 hemagglutinin proteins

Highly pathogenic avian influenza H5N1 viruses are capable of causing poultry epidemics and human mortality. 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: H5HA_148-162, H5HA _155-169, H5HA_253-267, H5HA_260-274, H5HA _267-281 and H5HA_309-323. Tetramer staining results confirmed that two immunodominant epitopes were DR1501-restricted: H5HA _155-169 and H5HA_267-281. 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.