Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage‐displayed hexapeptide library, and virus‐neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C‐terminal segment of the α2 domain of the heavy chain of human MHC class I molecules (MHC‐I α2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST‐fused MHC‐I α2‐ and FNIII‐derived oligopeptides interacted with recombinant fibers in a subgroup‐specific manner. In vivo, the MHC–I α2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In β2‐microglobulin‐transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4×1010/M. The results suggested that the conserved region of MHC‐I α2‐domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors.

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins ␣ v ␤ 5 and ␣ v ␤ 3 , which facilitate entry. The ␣ v integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing ␣ M ␤ 2 integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing ␣ v ␤ 5 and ␣ v ␤ 3 integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by ␣ v ␤ 5 , though transduction can be CAR and ␣ v ␤ 3/5 independent but is ␣ M ␤ 2 , MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.