Although, there appears to be substantial growth kinetic and thermostability differences between Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (Figures 4 and 5 ); there appears to be no significant difference between cellular Gag immune response generated from vaccination with Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (Figures 6 and 7) . Most importantly, vaccination with hexon-modified vectors in this study resulted in a humoral anti-HIV response. This is noteworthy, because HVR2 has not been fully explored for 'antigen capsid-incorporation' strategies. Anti-MPER humoral responses could be boosted after homologous Ad vector administration. At 40 d.p.p the MPER response in mice, vaccinated with hexon-modified vectors was approximately from 6 to 7 times higher than when using AdCMVGag, AdCMVEnv, or Ad5 vector. This is a critical finding of our study. It appears that vaccination with AdCMVEnv does not yield an antibody response. This finding could be explained in various ways: (1) since the HIV Env protein is a precursor protein that is catalyzed by protease to give the final product gp41 and gp120, it is possible that the production of gp120 is masking the antibody recognition of gp41 [37] ; (2) this finding might indicate that the antigen capsidincorporation strategy is superior to antigen transgene expression with regards to generation an in vivo immune response to MPER and/or other antigens [14] . In order to confirm the validity of the results obtained from all vaccination groups, a separate set of ELISA experiments were performed whereby sera from all vaccinated mice were bound to unmodified Ad vectors. All of the sera from the vaccinated groups bound unmodified wild type Ad5 equally well, indicating equal vaccination by Ad5, Ad-CMVGag, AdCMVEnv, Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15DE1 (data not shown). In addition, anti-MPER IgG1 and IgG2b isotype-specific responses were observed in mice vaccinated with the MPER capsid-modified vectors ( Figure 7) . Similar results were seen with IgG2a (data not shown), however; no MPER IgA-specific response was observed (data not shown). We did not expect to find much of the MPER IgA-specific immune response after vaccination with these vectors due to the route of vector administration. However, we plan to further investigate IgA-specific response in the context of targeted hexonmodified vectors in combination with alternative routes of administration. From a humoral immunity standpoint, IgA2 subclasses are reduced in saliva upon HIV infection, and total secretory IgA levels are reduced at later disease stages. Salivary IgA can be neutralizing to HIV-1 and HIV-2, and can block epithelial transmigration. In this instance, salivary IgA can be functional against HIV [38] [39] [40] . Figure 8 , compares the infectivity of Ad5/HVR2-MPER-L15(Gag) to that of the AdCMVGag vector in the presence or absence anti-Ad5 NAb found in human ascites. Both vectors were equally neutralized in the presence of human ascites fluid. We expected that Ad5/HVR2-MPER-L15(Gag) would have been neutralized to a lesser degree than AdCMVGag in the presence of human ascites. However, since this was not the case, we speculate that the polyclonal anti-Ad5 antibodies found in human ascites fluid is not masked by the addition of a single epitope incorporated within the hexon region. We also speculate that we might have observed different results with this assay if we had used monoclonal antibody, targeted to the hexon region where the insert was incorporated. If a monoclonal antibody was used in the presence of ascites fluid, Ad5/HVR2-MPER-L15(Gag) might escape neutralization largely as compared to AdCMVGag. However, this possibility was not explored further at this time because it does not diminish the clinical benefit of these vectors. Importantly, the Ad5/HVR2-MPER-L15(Gag) vector allowed boosting of Gag transgene production, as compared to the wild type Gag expressing vector (Figure 9 ), this result was statistically significant at p,0.03. This is an important finding for two reasons: