Fig. 1

Vaccine design and vaccine characterization. a Schematic representation of Ad5 vectors encoding either SIVmac239 gag or HIV-1 CON B gag, P2A preceded by a glycine-serine-glycine linker (not noted in the figure), and SIVmac239 env. The inserted antigens were flanked by huCMV and a SV40 polyadenylation signal. b Phylogenetic tree of Gag used in the Ad5 vaccines and for analysis. The distance values show the number of substitutions as a proportion of the length of the alignment (here excluding gaps). Upper panel is analysis of Gag and lower panel is analysis of p24 CE. Mu murine leukemia virus, used as an outlier; H HIV-1 CON B Gag; M SIVmac239 Gag; V SIVagm vervet Gag. c Gp160 and gp120 were detected in ultra centrifuge purified VLPs from the supernatants of Ad5 infected vero cells by SDS-PAGE (reduced) followed by western blot. SIVmac251 specific mAb KK46 was used for detection of gp120. Pr55 Gag and pr55 Gag cleavage products were detected using 183-5C-H12 HIV-1 p24 specific mAb. SIVmac239 gp130 and SIVmac251 BK28 pr55 Gag served as positive controls for gp120 and pr55 respectively. c is control lane. d Surface staining of vero cells infected with Ad5 vaccines (50 PFU/cell, 25 µg/ml mAb) or uninfected with indicated mAbs. e Ratio of AdHgSe to AdSgSe mAb binding calculated as mean fluorescence intensity × positively gated fraction. Calculations were performed on cells infected with 50 PFU/cell of either vaccine as in d, using 25 or 2.5 µg/ml mAb concentration. Shown are calculated ratios from two independent assays with the mean marked by horizontal lines