RGD inclusion in the hexon monomer provides adenovirus type 5-based vectors with a fiber knob-independent pathway for infection

Abstract

Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine "isogenic" viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An alphav-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands.

FIG. 1

Modification of the Ad5 hexon monomer by recombinational cloning in E. coli. (A) Shuttle plasmid IE27 (the detailed construction strategy is available upon request) is a kanamycin (Km)-selectable plasmid in which the xylE phenotypic marker has been inserted in place of HVR5 of Ad5 (residues 268 to 282). Its replication requires the polA gene product of E. coli. SacB is a suicide gene for E. coli in the presence of sucrose as the carbon source. a and b refer to HVR5-flanking sequences from Ad5 (numbers in parentheses refer to hexon amino acid numbering). (B) The xylE phenotypic marker of shuttle plasmid IE27 is bordered by unique NruI and Bsu36I sites to facilitate subsequent HVR5 modifications. (C) Principle of HVR5 modification by homologous recombination in E. coli (adapted from reference 5). Homologous recombination (HR) between identical sequences (black bars a and b) from a suicide ColE1 shuttle plasmid (e.g., IE27) and an RK2-derived (replication is polA independent) plasmid (e.g., AE18c) in a polA mutant of E. coli generates a kanamycin (Km)- and tetracycline (Tet)-selectable cointegrate. Resolution of the cointegrate by HR leads to the loss of the sacB suicide gene, which is selected by using sucrose as a carbon source. Depending on the recombination pathway, resolution of the cointegrate generates either the starting backbone AE18c or the xylE-containing IE27c backbone, which can be differentiated upon catechol addition (26). IE27c has been used as a recipient backbone to generate all HVR5-modified backbones and viruses used in this study (see the text).

FIG. 2

(A) C3 MAb-mediated virus immunoprecipitation. HVR5-modified Ads were immunoprecipitated under nondenaturing conditions before being subjected to Western analysis with a polyclonal serum against Ad5 (see the text). In lane C, 10⁹ VP of control AE18 virus was boiled for 2 min before being loaded. Lanes 1 to 10 correspond to AdIE30, AdIE35, AdIE37, AdIE39, AdIE40, AdIE43, AdIE44, AdIE45, AdIE46, and AdIE47, respectively. Arrows a, b, and c indicate the positions of the hexon, pV, and pVI/pVII polypeptides, respectively. (B) Virus opsonization inhibits lacZ transduction of W162 reporter cells. Control virus AE18 (lane C) or the indicated HVR5-modified viruses were incubated for 1 h at 37°C in the absence (solid bars) or presence (open bars) of C3 MAb before being used for infection. The number of X-Gal-positive cells was determined after 48 h.

FIG. 3

RGD inclusion in HVR5 enhances the transduction of cells naturally refractory to Ad5 infection. (A) Human primary aortic smooth muscle cells (Clonetics, San Diego, Calif.) were infected with control AE18 (top) or the RGD-containing virus AE57 (bottom) at an MOI of 1,000 or 10,000 VP/cell as indicated. X-Gal staining was carried out 48 h post-infection. (B) lacZ specific activity following infection with AE18 (□) or AE57 (●) at the indicated MOI. Extracts were prepared 48 h postinfection, at which time total protein and β-galactosidase activity were quantified. Data represent the means and standard deviations of duplicate experiments.

FIG. 3

RGD inclusion in HVR5 enhances the transduction of cells naturally refractory to Ad5 infection. (A) Human primary aortic smooth muscle cells (Clonetics, San Diego, Calif.) were infected with control AE18 (top) or the RGD-containing virus AE57 (bottom) at an MOI of 1,000 or 10,000 VP/cell as indicated. X-Gal staining was carried out 48 h post-infection. (B) lacZ specific activity following infection with AE18 (□) or AE57 (●) at the indicated MOI. Extracts were prepared 48 h postinfection, at which time total protein and β-galactosidase activity were quantified. Data represent the means and standard deviations of duplicate experiments.

FIG. 4

The RGD virus can use a fiber knob-independent pathway for infection. (A) 293 cells incubated without (left) or with (right) 100 μg of recombinant Ad5 knob per ml were subsequently exposed for 1 h to virus AE18 (top) or AE57 (bottom) at an MOI of 200 VP/cell. Unbound virus was then removed, fresh medium was added, and the cells were stained with X-Gal after 24 h. (B) Same as for panel A, except that the lacZ-specific activity in the cellular extracts was quantified in the absence (open bars) or presence (solid bars) of recombinant knob. Data represent the means and standard deviations of duplicate experiments.

FIG. 4

The RGD virus can use a fiber knob-independent pathway for infection. (A) 293 cells incubated without (left) or with (right) 100 μg of recombinant Ad5 knob per ml were subsequently exposed for 1 h to virus AE18 (top) or AE57 (bottom) at an MOI of 200 VP/cell. Unbound virus was then removed, fresh medium was added, and the cells were stained with X-Gal after 24 h. (B) Same as for panel A, except that the lacZ-specific activity in the cellular extracts was quantified in the absence (open bars) or presence (solid bars) of recombinant knob. Data represent the means and standard deviations of duplicate experiments.