Fig. 6: Effect of pre-existing immunity on the immunogenicity and protective ability of DelNS1-H5N1 vaccine. | Nature Communications

Fig. 6: Effect of pre-existing immunity on the immunogenicity and protective ability of DelNS1-H5N1 vaccine.

From: Intranasal influenza virus-vectored vaccine offers protection against clade 2.3.4.4b H5N1 infection in small animal models

Fig. 6

A Schedule of immunization, serum collection, T cell response, virus challenge and sacrifice for BALB/c mice. Mice were intranasally vaccinated with H1N1/H3N2 mix (106 pfu mixture of H3N2 and H1N1 DelNS1 LAIVs (1:1), n = 23), or mock-vaccinated (n = 8). 4 weeks after immunization, 15 mice from H1N1/H3N2 mix group and 8 mice from mock group were vaccinated with DelNS1-mH5N1 (106 pfu). Mice were challenged with mink H5N1 virus (104 pfu) 4 weeks later. B Serum was collected at week 3 post H1N1/H3N2 mix immunization and tested for IgG titers against H3N2 and H1N1 DelNS1 LAIVs and micro-neutralization titers against live viruses (n = 8 each group). BAL IgA level against H1N1 DelNS1 LAIVs were measured 8 weeks after H1N1/H3N2 mix vaccination (n = 8 each group). C Serum were collected 3 weeks after DelNS1-mH5N1 immunization and tested for IgG titers against live virus or H5N1-HA1 protein and micro-neutralization titers against live virus (n = 8 each group). D 8 weeks after prime immunization, for the H1N1/H3N2 mix group and H1N1/H3N2 mix + DelNS1-mH5N1 group (n = 6), PE-Cy5 CD45 antibody was injected intravenously 5 min before sacrifice. Lung cells and splenocytes were stained with Zombie, anti-CD8 and NP147 tetramer. The frequency of IV-CD8 + NP147 tetramer out of all live IV-CD8 T cells was displayed. Lung cells and splenocytes were stimulated with NP147 and stained with surface markers, followed by fixation, permeabilization, and intracellular staining for IFN-γ. The frequency of tissue-resident memory cells in lungs (IV-IFNγ+CD69+CD103+ CD8+ T cells out of all live IV-CD8 T cells) and spleens (IV-IFNγ+CD8+ T cells out of all live IV-CD8 T cells) was displayed. E 8 weeks after prime immunization, mice in H1N1/H3N2 mix + DelNS1-mH5N1 group (n = 9) and mock group (n = 8, two independent experiments) were challenged with mink H5N1 virus (104 pfu); body weights were monitored for 14 days, and viral titers in lungs and NT were measured at 4 dpi. LOD: limit of detection. Data represent mean values ± SD of results. Statistical analysis was performed using Student’s t-test (two-tailed) (B, D, E) and one-way ANOVA (C). The mouse cartoon was created in BioRender. Wang, P. (2025) https://BioRender.com/n83i107. Source data are provided as a Source Data file.

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