Figure 1.

HD-Ad-FS induced efficient systemic and mucosal immunities in BALB/c mice. (A) Schematic of HD-Ad-FS vaccine. ITR, inverted terminal repeats; Ψ, adenoviral packaging signal; CBA, promoter of chicken beta-actin gene; UbC intron, first intron of ubiquitin C gene; FS, full-length spike gene of SARS-CoV-2 ancestral strain; bGH PolyA, polyadenylation tail of bovine growth hormone gene. (B) Western blot analysis of FS protein expression. IB3-1 cells were transduced with HD-Ad-FS at the indicated MOI, and proteins were harvested at day 3 post-transduction. (C) Schematic timelines of BALB/c mouse experiments. BALB/c mice were intranasally administered with HD-Ad-FS or HD-Ad-control at single-dose (5x10⁹ vp) and prime-boost regimens (5x10⁹ + 5x10⁹ vp, three weeks interval). Three weeks after the last administration, animal samples were harvested. (D) Levels of FS-specific sIgA responses in BALs were determined with Enzyme-linked immunosorbent assay (ELISA). The starting dilution factor was 1:1. (E, F) Neutralization of live SARS-CoV-2 and VOCs infection by sera (E) and BALs (F). The Omicron used in this study was BA.1.18. The starting dilution factors were 1:50 and 1:5 for sera and BALs, respectively. (G) CD4⁺ T cell responses in the lungs of single-dose mice were measured by the production of IFN-γ (left), TNF (middle), and double-positive IFN-γ and TNF (right) at 3 weeks post-immunization, following ex vivo stimulation with spike antigen. (H) The production of IFN-γ (left), TNF (middle), and double-positive IFN-γ and TNF (right) in CD4⁺ T cells in the spleens of single-dose mice were measured at 3 weeks post-immunization, following ex vivo stimulation with spike antigen. (I) IFN-γ producing CD8⁺ T cells in the lungs of single-dose mice were measured by ex vivo stimulation with spike antigen. In all figures, dots represent individual mice (n=4 or 5). The dotted lines represent the limit of detection (LOD) of the assays. Statistical analyses were performed by one-way ANOVA. Bars and errors represent the geometric mean with geometric SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.