Fig. 4: Protection of HLA-A*02:01/DR transgenic mice from MPXV infection by dual immunization with MPX-Mix antigens.

a Schematic diagram of the dual immunization design using Mix-12 and MPX-EPs antigens. HLA-A*02:01/DR transgenic mice were immunized with MPX-m-Mix (containing 3 μg MPX-EPs and 12 μg Mix-12 per mouse), MPX-p-Mix (containing 12 μg proteins from Mix-12 and 3 μg peptides from MPX-EPs per mouse), or PBS with 8.7% sucrose as a control. A booster vaccination was administered 3 weeks later. Blood and lymph nodes were collected 1-week post-booster for humoral immune response analysis, while cells from lungs and spleens were collected 3 weeks post-booster for cellular immune response evaluation. Immunized mice (n = 5 per group) were challenged with MPXV virus (1.0 × 10⁷ PFU) 3 weeks after the booster vaccination. b, c Antibody production following immunization against MPXV. MPXV-specific IgG antibodies in the sera collected 21 days after the booster vaccination were detected with ELISA. Neutralizing antibody titers (NT₅₀) were determined by plaque reduction neutralization test. d–f Th1-biased cellular immune response induced by MPX-Mix. Splenocytes from immunized HLA-A*02:01/DR1 transgenic mice (n = 5) collected 21 days post-booster immunization were re-stimulated ex vivo and subjected to IFN-γ ELISpot (d) (lower limit of detection (LLOD) = 20) and IL-4 ELISpot (e) (LLOD = 2), with the correlation of IL-4- and IFN-γ-secreting cells shown in the scatter plot (f). g–h Analysis of lung-resident CD8⁺ T cells from immunized mice (n = 5 per group). Lung cells were stained with anti-CD45-Alexa Fluor™ 700, CD8-PerCP-Cyanine5.5, CD69-FITC, and CD103-AF594. The frequencies of CD45⁺CD8⁺CD69⁺CD103⁺ T cells were measured by flow cytometry, with the representative flow cytometry plots presented. i–k Protection conferred by MPX-m-Mix or MPX-p-Mix against MPXV. Viral loads in the lungs (i) and lung pathology (j, k) were evaluated at 4 days post MPXV inoculation (i.n., 1.0 × 10⁷ PFU, n = 5 mice per group), Scale bar is 200 μm. Data are representative of two independent experiments with three technical replicates (b–k). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons (n = 5). Data are presented as mean ± SEM (b–e, h, I, k). Source data are provided as a Source Data file.