Fig. 3: DelNS1-H5N1 vaccines induce CD4+ and CD8+ T cell responses.

A Schedule for immunization of BALB/c mice. At week 4 after immunization, 2 μg of PE-Cy5 conjugated CD45-specific antibody was injected intravenously via the tail vein 5 min before sacrifice (n = 8 for each group). B Lung cells and splenocytes were obtained and stained with Zombie, anti-CD8 and NP₁₄₇ tetramer. Flow cytometry gating was performed to assess the frequency of live DelNS1-cH5N1-induced non-circulating (IV CD45-PE-Cy5 negative) NP-specific CD8+ T cells among lung cells and splenocytes (percentage IV-CD8 + NP₁₄₇ tetramer+ out of all live IV-CD8+ T cells). C Lung or spleen cells were stimulated with NP₁₄₇ or NP₅₅ peptide overnight. Surface markers (CD69, CD103, CD4, CD8 and Zombie) were stained, followed by fixation, permeabilization, and intracellular staining for IFN-γ. DelNS1-cH5N1 induced tissue-resident memory cells in lungs and spleens were displayed (IV-IFNγ + CD69 + CD103 + CD8 + T cells and IV-IFNγ+CD69+CD4+ T cells out a percentage of all live IV- CD8+ and CD4+ T cells, respectively). D DelNS1-mH5N1 induced non-circulating T cells in lung cells and spleens (IV-CD8 + NP₁₄₇ tetramer out of all live IV-CD8 T cells). E DelNS1-mH5N1 induced tissue-resident memory cells in lungs and spleen were displayed (IV-IFNγ+CD69+ CD103+CD8+ T cells and IV-IFNγ + CD69+ CD4+ T cells out a percentage of all live IV- CD8+ and CD4 + T cells, respectively). Data represents the mean values ± SD of results. Statistical comparisons between means were performed by Student’s t-test (two-tailed). The mouse cartoon was created in BioRender. Wang, P. (2025) https://BioRender.com/n83i107. Source data are provided as a Source Data file.