Construction and validation of four versions of hAd5-based V3 vectors. A) Construction schematic of four versions of hAd5-based V3 vectors. Four V3 antigens (His-V3, V3, long-V3 and V3-His) were genetically inserted into the HVR1 locale of Ad hexon, using the strategy of “Antigen Capsid-Incorporation”. The E1 region of all four vectors was replaced with GFP-Luc. B) After the vector construction and rescue, four vectors were validated for His presentation via western-blot analysis, with the use of anti-penta-His MAb. Line 1, Ad-HVR1-lgs-His6-V3; line 2, Ad-HVR1-V3; line 3, Ad-HVR1-long-V3 and line 4, Ad-HVR1-lgs-V3-His6-lgs. C) V3 presentation was also validated via western-blot analysis, with the use of anti-gp120 (902) MAb. The vector order was the same as the arrangement in Figure 1B. D) Anti-IIIB-V3-21 MAb was also used to validate the V3 presentation in the vectors. The vector order was the same as the arrangement in Figure 1B.