Identification of Sites in Adenovirus Hexon for Foreign Peptide Incorporation

Abstract

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His 6 ) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His 6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His 6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His 6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His 6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His 6 epitopes did not appear to mediate His 6 -dependent viral infection, as assessed in two His 6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

“…The hypervariable region 5 (HVR5) surface loop of hexon is another attractive site for possible ligand incorporation because of its sheer abundance (720 copies) in the Ad virion. With the exception of the 71 amino acid BAP from P. shermanii , this site has only been shown to accommodate small insertions like RGD, His 6 , and other epitopes [Crompton et al, 1994, Vigne et al, 1999, Wu et al, 2005.…”

“…Detailed structural data on the major capsid proteins [Rux & Burnett, 2000,Xia et al, 1994 have greatly facilitated the genetic incorporation of foreign peptides and proteins into exposed regions of the Ad capsid. Initial work demonstrated that short peptides and epitopes could successfully be grafted into the surface exposed HI loop ] and C-terminus [Wickham et al, 1996] of the fiber knob domain, the RGD-containing loop of penton base [Wickham et al, 1997], the hypervariable region 5 (HVR5) loop of the hexon [Crompton et al, 1994,Wu et al, 2005, and the C-terminus of protein IX .…”

Characterization of a Permissive Epitope Insertion Site in Adenovirus Hexon

“…Subsequent work has verified that a hexahistidine tag can be inserted into HVR2, HVR3, HVR5, HVR6, and HVR7 without compromising virus viability (66). Initial studies attempting to take advantage of the malleability of HVRs were primarily focused on inserting short sequences to attain vector retargeting (63).…”

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues

“…Further experiments revealed the possibility of expanding the vector tropism by inserting a heparinbinding domain (Wickham et al, 1996), or an Arg-Gly-Asp (RGD)-containing peptide into the fiber knob (Dmitriev et al, 1998). Although different studies indicated the feasibility to target vectors to specific cell types, genetic modification often resulted in failure to rescue viable viruses, or in an impaired virus packing, peptide exposure, and vector transduction (Leissner et al, 2001;Wu et al, 2005). Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, use of random peptide libraries displayed directly on the AdV capsid allows isolation of viable vectors with high affinity for specific tissues or cells (Miura et al, 2007).…”