The incorporation of the HIV-1 epitope into the hexon protein of Ad5 may interfere with the viral infectivity and/or reduce recognition by anti-Ad5 neutralizing antibodies (NAb). We therefore compared infectivity of Ad5/HVR2-MPER-L15(Gag) to that of the original AdCMVGag virus in the presence or absence of anti-Ad5 NAb found in human ascites that have been described previously [36] . In the absence of ascites, the frequency of cells expressing Gag after infection with AdCMVGag or Ad5/HVR2-MPER-L15(Gag) was 81.8% and 80.6% respectively, indicating that the incorporation of HIV-1 epitope into the hexon protein of the Ad5 vector did not reduce virus infectivity ( Figure 8A ). Compared to the unmodified Ad5 vector, the presence of the HIV-1 epitope did not alter neutralization by anti-Ad5 NAb present in the human ascites ( Figure 8B ). Therefore the HIV-1 epitope did not facilitate escape from pre-existing anti-adenoviral humoral immune response, probably due to the polyclonal nature of the ascites NAbs. for ELISA isotype binding assays. 10 mM of purified MPER (EKNEKELLELDKWASLWNWFDITN) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera followed by isotype specific antibodies. The binding was detected with HRP conjugated secondary. OD at 405 nm represents of isotype-specific MPER antibody levels in sera. doi:10.1371/journal.pone.0011815.g007