Fig. 6.

Loss of Nef Immunogenicity when Nef is expressed as part of GagPol or Pol fusion proteins. (A) The Pol-NTV (plasmid 35S) and the CATE-Pol-NTV (plasmid 44S) fusion plasmids were expressed in human 293 cells upon transfection of 100 ng of DNA. Two days later the cell extracts were analyzed on Western immunoblot using pooled plasma from SIVmac251 infected macaques. The relative GFP relative values (× 100) shown in lanes 1-2 were 570 and 570, respectively. (B) The immunogenicity of the pol-NTV fusion proteins. Balb/c mice (N=6) were vaccinated twice with 100 μg of DNA at day 0 and week 4, and splenocytes were prepared 2 weeks later. Cellular immune responses to pol, Nef, Tat, and Vif peptide pools were analyzed by ELISPOT. The mean and SEM are shown. (C) The GagPol fusion protein contains Gag (aa 1-439) artificially linked (via 13 aa of the ORF preceding Pol) to Pol (aa 1-951) (plasmid 82S). Nef was fused to the C-term (plasmid 179S) or the N-term (plasmid 185S) of GagPol. The plasmids expressing the fusion proteins were expressed in human 293 cells and analyzed on Western immunoblot probed with pooled plasma from SIVmac251 infected macaques. Plasmids expressing only Gag (plasmid 10S; non-myristoylated gag), Pol (plasmid 88S) and Nef (plasmid 180S) are shown. The relative GFP values (× 100) of the transfections shown in lanes 1-6 were: 140, 150, 150, 150, 150 and 140. (D) The immunogenicity of the indicated GagPol fusion proteins was tested. Balb/c mice (N=6) were vaccinated with 100 μg twice at day 0 and week 4, and splenocytes were prepared 2 weeks later. Cellular immune responses to Gag, Pol, and Nef peptide pools were analyzed by ELISPOT. The mean and SEM are shown.