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 αv-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.

Adenoviral vector-based strategies against infectious disease and cancer

“…138 The penton base and pIX were tested for antigen-incorporation in some studies. [139][140][141][142] However, compared with the other three proteins on the capsids, the penton base was rarely incorporated with foreign antigens perhaps due to the structural constraints.…”

Adenovirus receptors and their implications in gene delivery

“…Incorporation of RGD or polylysine (pK7) ligands on these locations led to enhancement of Ad infectivity to a wide variety of target cells (for example tumors cells and DCs) that overexpress integrins or HSPGs, respectively (Dmitriev et al, 1998;Koizumi et al, 2003;Wickham et al, 1996;Wu et al, 2002). Besides fiber knob, other capsid proteins such as hexon, penton, pIX or pIII have also been investigated to alter the vector tropism by cell-specific ligand incorporation Glasgow et al, 2006;Vellinga et al, 2004;Vigne et al, 1999;Wu et al, 2005). Utilizing affibodies or library screening approaches, cell-specific targeting ligands can be identified that can be incorporated to Ad capsid to form stable virions (Belousova et al, 2008;Henning et al, 2002;Nord et al, 1997).…”

Strategies to overcome host immunity to adenovirus vectors in vaccine development

“…The discovery that the majority of Ad5 NAbs are directed against Ad hexon HVRs [97], and confirmation that the HVRs of Ad5 could be engineered to incorporate foreign peptides, provided the rationale for the developing chimeric Ad5 vectors that express the HVRs of other serotypes [134][135][136]. Chimeric Ad5 vectors expressing the HVRs from Ad48 (Ad5HVR48) induced significantly greater immune responses to the transgene product than unmodified Ad5 vectors did in both mice and rhesus monkeys with Ad5 PEI [137].…”