Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid

Abstract

Recombinant adenoviruses are frequently used as gene transfer vehicles for therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors. Single-chain antibodies have a great potential to achieve such cell type specificity. In this study, we evaluated the efficiency of incorporation of a single-chain antibody fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end, the codons for the single-chain antibody fragments (scFv) 13R4 were fused with those encoding of pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against beta-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted human adenovirus-5 vectors on these cells, the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand beta-galactosidase. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.

Figure 1

A. Schematic representation of the pIX.flag.75.13R4.MYC.HIS fusion protein exposing the 13R4 scFv above the hexon capsomers. B. Schematic representation of the lentiviral system. The lentiviral vectors used in this study are so-called SIN vectors, and contain the Rev-responsive element sequence, the central poly-purine tract (cPPT), , , and the human hepatitis B virus-derived posttranscriptional regulatory element. The encephalomyocarditis virus internal ribosomal entry site (IRES) was obtained from pTM3, the NPTII coding region was isolated from peGFPn2 (Clontech, BD Biosciences, The Netherlands).

Figure 2

A. Immunohistochemistry assay for detection of the pIX.flag.75.13R4.MYC.HIS produced by the 911-pIX.flag.75.13R4.MYC.HIS cells. The production of pIX.flag.75.13R4.MYC.HIS was visualized using mouse anti-flag and FITC-labeled goat-anti-mouse antibodies. The nuclei were stained using propidium iodide. B. Western analysis of pIX.flag.75.13R4.MYC.HIS levels in the 911-pIX.flag.75.13R4.MYC.HIS cells. The pIX.flag.75.13R4.MYC.HIS amount in the complementing cell line 911-pIX.flag.75.13R4.MYC.HIS was compared with the pIX amounts during wt HAdV-5 infection. The Western analysis was performed using anti pIX serum. C. Western analysis of the incorporation efficiency of pIX.flag.75.13R4.MYC.HIS. To test the incorporation efficiency of pIX.flag.75.13R4.MYC.HIS produced by the 911-pIX.flag.75.13R4.MYC.HIS cells, HAdV-5ΔpIX.CMV.GFP/LUC was propagated on the cell line, purified by CsCl centrifugation, and protein lysates of the purified viruses sample were made for Western analysis. The amount of the pIX fusion proteins in HAdV-5ΔpIX.CMV.GFP/LUC propagated on 911-pIX.flag.75.13R4.MYC.HIS was compared with wt HAdV-5, with anti-pIX serum and, as a virus-particle loading control, the 4D2 antibody directed against the fiber protein. D. Immunoelectron microscopic analysis of HAdV-5ΔpIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS. To test the accessibility of the HIS epitope on viruses loaded with pIX.flag.75.13R4.MYC.HIS were bound on copper grids. The HIS epitope was detected with penta-HIS antibody, followed by rabbit anti-mouse immunoglobulin and gold-labeled Prot.A. E. Heat stability of HAdV-5ΔpIX.CMV.GFP/LUC with pIX.flag.75.13R4.MYC.HIS in their capsid. HAdV-5ΔpIX.CMV.GFP/LUC was propagated on 911-pIX.flag.75.13R4.MYC.HIS cells. Similarly, HAdV-5ΔpIX.CMV.GFP/LUC and HAdV-5.CMV.GFP/LUC were propagated on 911 cells as negative and positive control, respectively. Freeze-thaw lysates were incubated at 45°C for various times. Residual infectious virus titers were estimated by determining the capacity of the virus to induce luciferase activity in U2OS cell 24 h after infection. The results are presented as percentages of residual luciferase activity. Each bar represents the cumulative mean ± standard deviation of triplicate analyses.

Figure 3

To test if the HAdV-5ΔpIX.CMV.GFP/LUC particles loaded with pIX.flag.75.13R4.MYC.HIS were able to bind specifically β-galactosidase on the outside of the virion, viruses were mixed with β-galactosidase and purified via the standard CsCl purification protocol. A. Western analysis. The captured β-galactosidase was detected using anti-β-galactosidase. Wt HAdV-5 was used as negative control. B. Immunoelectron microscopic analysis of β-galactosidase bound to HAdV-5ΔpIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS. The β-galactosidase was only detected on the viruses that carried the 13R4 scFv fused to pIX.

Figure 4

DAKO IDEIA β-galactosidase binding assay. To measure the binding capability of 13R4 on the surface of the virion to its native ligand β-galactosidase, the HAdV-5ΔpIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS were incubated with β-galactosidase and trapped on a DAKO IDEIA strips. The bound β-galactosidase was detected by measuring OD420 (left y-asis) after adding ONPG together with the Z-buffer (n=3). As negative control an identical number of wt HAdV-5 particles were used.