Fig. 1

Design of adenoviral vectors and characterization in vitro and in vivo. a Schematic representation of the adenoviral vectors used in the study: Ad-HIVB encodes HIV-1 consensus clade B (HIVconB) Gag, P2A preceded by a glycine-serine-glycine linker (GSG; not noted in the figure) and HIV-1 JR-FL (clade B) Env; Ad-SIV encodes SIVmac239 Gag and Env separated by GSG and P2A; Ad-Ii-SIVCErvv encodes mouse Ii aa 1–75 (Ii) fused to SIV CEs and SIVmac239 Rev, Vif and Vpr. b Vero cells were infected with 50 IFU/cell Ad-HIVB and were stained after 2 days with the monoclonal antibodies VRC01, PGT145 and PGT151 targeting HIV-1 Env (shown in dark grey). APC-labelled anti-human IgG Fc served as a secondary antibody and the cells were analyzed by flow cytometry. Uninfected stained cells served as a control (light grey). c Vero cells were infected with 50 IFU/cell Ad-HIVB and after 48 h VLPs were purified from the cell culture supernatant. The VLP samples were analyzed by SDS-PAGE followed by Western Blot, which was stained with an anti-P2A antibody. Cells infected with an Ad5 vector not encoding P2A (Ad-noP2A) served as a negative control. d CD1 mice were vaccinated with Ad-HIVB (n = 5) and the induced T-cell responses were analyzed 18 days later by intracellular cytokine staining of stimulated splenocytes. The peptide pools used for stimulation are noted on the X-axis. The total numbers of IFNγ+ CD8+ and CD4+ T-cells per spleen were measured by flow cytometry. Horizontal lines indicate the geometric mean and significant differences are marked by asterisks with *(p ≤ 0.05). e CD1 mice were immunized with Ad-Ii-SIVCErvv (n = 10). After 14 days CD8+ and CD4+ T-cell responses to the vaccine antigens were analyzed as in d