After establishing the technical feasibilities allowing us to place epitopes into HVR2 or HVR5 [16, 19] , we sought to explore whether we could present HIV antigens within the Ad protein hexon as well as express HIV antigens as a transgene within Ad5. The rationale for choosing a portion of the MPER (EKNEKEL-LELDKWASLWNWFDITN), derived from gp41 for incorporation into the Ad capsid is based on the fact that the gp41 envelope protein ectodomain is a target of three broadly neutralizing anti-HIV-1 antibodies [32] . Gp41 antigenic function is conformation dependent, therefore we planned to insert a linker sequence onto the MPER core region in order to have the MPER epitope presented on the Ad capsid as close to it's natural conformation as possible [33] . Our vaccine vector design also embodies the incorporation of the HIV Gag gene within the Ad DE1 region. The HIV Gag gene is a major structural protein of the HIV virus. The Gag protein has been frequently used for HIV vaccine schemes [34] . The DNA sequence corresponding to a 24 amino acid region of the MPER plus a 15 amino acid linker was amplified by PCR and then cloned into the hexon HVR2 shuttle vector as previously described [16] . In addition, the HIV Gag gene was subcloned into an DE1 shuttle vector under control of the CMV promoter and transferred into the E1-deleted Ad5 genome (Ad5/HVR2-MPER-L15(Gag)) by homologous recombination. In addition, a control vector was generated, which contained an E1-deleted Ad5 genome in combination with MPER within the hexon HVR2 region (Ad5/HVR2-MPER-L15DE1). A control Ad genome expressing the Gag gene under the CMV promoter with no hexon modification (AdCMVGag) was generated in this study as well. The resulting Ad genomes were partially sequenced to confirm that the correct genes were incorporated. Subsequent transfection of HEK293 cells with the resulting recombinant genomes resulted in rescue of the following vectors: AdCMVGag, Ad5/HVR2-MPER-L15DE1, and Ad5/ HVR2-MPER-L15(Gag). In order to further confirm vector identities, hexon and Gag-specific PCR analyses were performed using genomic DNA from the purified virions ( Figure 1A-B) . With regard to hexon-specific PCR, AdCMVGag was found to have a wild type hexon PCR profile producing ,450 base pairs (bp) PCR fragment using the hexon-specific primers designed to amplify a regions between HVR2 and HVR5 ( Figure 1A, lane 1) . A hexon specific PCR, on the Ad5/HVR2-MPER-L15(Gag) construct revealed a 500 bp fragment, suggesting the expected insertion ( Figure 1A, lane 2) . The addition of ,50 bp in Figure 1A , lane 2 indicates the incorporation of the MPER-L15 DNA within the hexon region. Both AdCMVGag and Ad5/ HVR2-MPER-L15(Gag) were found to be positive in a Gagspecific PCR assay, producing a ,1500 bp product with Gagspecific PCR primers ( Figure 1B, lanes 1-2, respectively) . A diagram depicting the vector construction of vectors used in this study is illustrated in Figure 1C . Expression and display of HIV antigens within adenoviral vectors After successful incorporation of HIV genes we next sought to verify expression of our transgene and capsid incorporation at the protein level by Western blot analysis. HEK293 cells were infected with unmodified Ad5 (as a negative control) or one of the HIV-Gag containing viruses (Ad5/HVR2-MPER-L15(Gag) or Ad-CMVGag, respectively. Cell lysates were prepared 24 hours post-infection and subjected to Western blot analysis with anti-Gag-specific antibody. Gag protein expression was evidenced by the presence of a ,55 kDa molecular weight protein band in the lysates from cells infected with either hexon-modified Gag expressing vector Ad5/HVR2-MPER-L15(Gag) (Figure 2 -A, lane 2) or the AdCMVGag (Figure 2-A, lane 3) . As expected no Gag protein expression was detected in the lysates from cells infected with the unmodified Ad5 virus (Figure 2-A, lane 1) .