Improved adenovirus type 5 vector-mediated transduction of resistant cells by piggybacking on coxsackie B-adenovirus receptor-pseudotyped baculovirus

Abstract

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.

FIG. 1.

Pseudotyping BV with human CAR glycoproteins. (a) Western blot analysis of control, parental (BV), and CAR-pseudotyped baculovirions (BV^CAR). BV (lane 1) and BV^CAR (lane 2) purified by ultracentrifugation were analyzed by SDS-PAGE and immunoblotting, using anti-gp64 and anti-CAR monoclonal antibodies and anti-mouse IgG conjugate. (b to g) Immuno-EM analysis. Virion samples deposited on grids were negatively stained with uranyl acetate and then reacted with monoclonal antibody against CAR, followed by anti-mouse antibody tagged with 20-nm colloidal gold. (b and c) General views of immunogold-stained BV^CAR preparations. Note the immunogold labeling associated with BV^CAR virions and the low level of background labeling. (d to g) Enlargement of anti-CAR gold-labeled BV^CAR virions. The number of grains associated per virion ranged from 0 (c and d) to 7 (b), with the highest frequency at 1, as shown in panels d and e.

FIG. 2.

EM and immuno-EM of BV^CAR. (A) Occurrence of BV^CAR-BV^CAR complexes. Samples of CAR-pseudotyped baculovirions (BV^CAR) were deposited on grids, negatively stained with uranyl acetate, and then reacted with monoclonal antibody against CAR, followed by anti-mouse antibody tagged with 20-nm colloidal gold, as in Fig. 1. Shown are spontaneously occurring pairwise (a to c) or multiple (d) associations of BV^CAR virions. (B) Anti-gp64 immunogold labeling of CAR-pseudotyped baculovirions. (a to c) BV^CAR virions deposited on grids were negatively stained with uranyl acetate and then reacted with monoclonal antibody against peplomer gp64, followed by anti-mouse antibody tagged with 20-nm colloidal gold grains. (d) Same reaction as in panels a to c performed on BV^CAR-Ad5GFP complexes deposited on grids. Note that gp64 and Ad5GFP virions are positioned at opposite poles of the baculovirion.

FIG. 3.

Immunogold labeling of BV^CAR. (a) Comparison of the labeling efficiency of BV^CAR samples using anti-gp64 or anti-CAR monoclonal antibodies. The number of anti-gp64 and anti-CAR gold grains was counted per BV^CAR particle, in a population of 70 to 100 virions. (b) Topology of gp64 and CAR molecules on the baculoviral envelope, as determined by immunogold labeling. The position of gold grains on BV^CAR virions was determined by measuring the distance of the center of the gold grain to the tip of the virion head. Results were expressed as map units, defined as the percentage of the BV total length, which was assigned the 100% value. Shown are the means of three separate experiments ± SEMs.

FIG. 4.

EM and immuno-EM of BV^CAR-Ad5GFP complexes. BV^CAR-Ad5GFP complexes deposited on grids were negatively stained with uranyl acetate and examined under the EM (a to c and g to i) or further incubated with anti-CAR monoclonal antibody and 20-nm colloidal gold-tagged anti-mouse antibody (d to f), in order to test the anti-CAR reactivity of the BV^CAR-Ad5GFP complex. Ad5GFP virions are marked with asterisks. Panels a to d show BV^CAR virions associated with a single particle of Ad5GFP, whereas panels e to g show BV^CAR virions associated with two Ad5GFP particles. Panels b and c are enlargements of BV^CAR-Ad5GFP complexes showing filamentous structures connecting adenovirions to the baculoviral envelope (arrows). Note that CAR molecules were not all occupied by Ad5GFP virions, since anti-CAR antibodies still reacted with the complexes (d to f). Panels h and i show Ad5GFP virions bridging two BV^CAR virions.

FIG. 5.

Transduction of CAR-negative (CHO) or CAR-positive (CHO-CAR) cells by BV^CAR-Ad5GFP complex. (A) Fluorescent microscopy. CHO cells were transduced by Ad5GFP alone (MOI of 100 vp/cell) (a), BV^CARGFP alone (1,000 vp/cell) (b), or BV^CAR-Ad5GFP complex (Ad5GFP MOI of 100 vp/cell; Ad5GFP/BV^CAR vp ratio of 1:10) (c). (B) Flow cytometry. Bar graph representation of the efficiency of transduction of CHO (a) or CHO-CAR (b) by Ad5GFP alone (open bars) or BV^CAR-Ad5GFP complex (solid bars). Complexes were generated by mixing a constant amount of BV^CAR (corresponding to 500 vp/cell) with increasing amounts of Ad5GFP, as indicated on the x axis. (C) Transduction efficiency of CHO cells by control vector BV^CARGFP. Flow cytometry analysis of CHO cells (gray bars) or CHO-CAR cells (black bars) transduced by BV^CARGFP alone at increasing MOIs, as indicated on the x axis. Results, expressed as the percentages of GFP-positive cells, represent the means of three separate experiments ± SEMs. *, P < 0.05; **, P < 0.01; ns, no significant difference.

FIG. 6.

Transduction of CAR-negative human cell lines by BV^CAR-Ad5GFP complex. (A) Nontumor cells. Fluorescent microscopy of MM39 cells transduced by Ad5GFP alone (MOI of 20 vp/cell) (a), BV^CARGFP alone (500 vp/cell) (b), or BV^CAR-Ad5GFP complex (Ad5GFP MOI of 20 vp/cell; Ad5GFP/BV^CAR vp ratio of 1:25) (c). (B) Tumor cells. Flow cytometry analysis of human cancer cell lines RD, SKOV3, and SKBR3 transduced by Ad5GFP alone (20 vp/cell), BV^CARGFP alone (500 vp/cell), or BV^CAR-Ad5GFP complex (Ad5GFP MOI of 20 vp/cell; Ad5GFP/BV^CAR vp ratio of 1:25). Results, expressed as the percentages of GFP-positive cells, represent the means of three separate experiments ± SEMs. *, P < 0.05; **, P < 0.01; ns, no significant difference.

FIG. 7.

Transduction of CAR-negative human primary cells by BV^CAR-Ad5GFP complex. Graphs on left show flow cytometry results. Bar graph representation of the efficiency of gene transfer mediated by Ad5GFP alone versus BV^CAR-Ad5GFP complex in different human primary cells, as indicated above each panel. Cells were transduced by Ad5GFP alone at increasing MOIs or by BV^CAR-Ad5GFP complexes at a constant MOI of BV^CAR (500 vp/cell) and increasing MOIs of Ad5GFP, as indicated on the x axis. Results, expressed as the percentages of GFP-positive cells, represent the means of three separate experiments ± SEMs. The black bars on the far left of the graphs represent the values obtained with the control baculoviral vector BV^CARGFP alone, at an MOI of 500 vp/cell. The right panels show fluorescent microscopy results. Shown are cell samples transduced at the maximal infectivity of each separate vector or biviral complex, as indicated above each panel. Cells were transduced by Ad5GFP alone (left), BV^CARGFP alone (middle), or BV^CAR-Ad5GFP complex (right). *, P < 0.05; **, P < 0.01; ***, P < 0.005.

FIG. 8.

Influence of BV^CAR-to-Ad5GFP ratios on BV^CAR-Ad5GFP-mediated transduction of human primary cells. (a) Dermal fibroblasts and synoviocytes were transduced by BV^CAR-Ad5GFP complex generated using a constant MOI of BV^CAR (500 vp/cell) and various MOIs of Ad5GFP, as indicated on the x axis. (b) Cells were transduced by BV^CAR-Ad5GFP complex generated using a constant MOI of Ad5GFP (20 vp/cell) and various MOIs of BV^CAR, as indicated on the x axis. (c) Cell transduction efficiency by BV^CAR-Ad5GFP complex was evaluated using a wide range of BV^CAR-to-Ad5GFP ratios. The transduction efficiency was expressed as the percentage of GFP-positive cells, assayed by flow cytometry (means of three separate experiments ± SEMs).

FIG. 9.

Role of CAR and fiber knob in BV^CAR-Ad5GFP-mediated cell transduction. (a) Requirement for the fiber knob domain in Ad5GFP vector. Human dermal fibroblasts were transduced by Ad5GFP-R7ΔKnob alone at increasing MOIs or a mixture of BV^CAR at a constant MOI (500 vp/cell) and Ad5GFP-R7ΔKnob at increasing MOIs, as indicated on the x axis. (b) Requirement for CAR glycoprotein on the baculoviral membrane. Human dermal fibroblasts and synoviocytes were transduced by a mixture of Ad5GFP and BV^CAR or a mixture of Ad5GFP and nonpseudotyped BV (parental AcMNPV empty vector) at a constant MOI of Ad5GFP (10 vp/cell) and various BV or BV^CAR inputs at MOIs of 0, 250, and 500 vp/cell. (c and d) Requirement for CAR-fiber interaction. Human dermal fibroblasts were transduced by a mixture of BV^CAR (MOI of 250 vp/cell) and Ad5GFP (20 vp/cell), containing anti-CAR monoclonal antibody added at different dilutions. In panel c, anti-CAR antibody was added after complex formation, by premixing the two viruses followed by incubation for 1 h at 37°C (“post”). In panel d, anti-CAR antibody was added simultaneously with both viruses before complex formation (“pre”). Virus samples and antibody were further incubated for 1 h at 37°C. Controls consisted of Ad5GFP samples at the same MOI incubated with the same antibody dilutions. Results were expressed as the percentages of GFP-positive cells, assayed by flow cytometry (means of three separate experiments ± SEMs). **, P < 0.01; ns, no significant difference.

FIG. 10.

Mechanism of cellular uptake of the BV^CAR-Ad5GFP complex. (a and b) Cell binding kinetics. Ad5GFP alone (open symbols), BV^CAR alone (open symbols), or BV^CAR-Ad5GFP complex (filled symbols) was incubated with CHO cells at 37°C for 50 min, and cell samples were withdrawn every 10 min p.i. After washing, cell-associated virions were assayed in cell lysates using real-time quantitative PCR. Results were expressed as the numbers of adenoviral and baculoviral genomes recovered per cell, using the beta-actin gene as an internal control. (a) Ad5GFP genomes: Ad5GFP alone (y = 0.24x, R² = 0.993) and BV^CAR-Ad5GFP complex (y = 0.49x, R² = 0.977). (b) BV^CAR genomes (y = 10.5x, R² = 0.91). (c) Role of baculoviral gp64. BV^CAR-Ad5GFP complex, at the BV^CAR/Ad5GFP MOI ratio of 500:20 vp/cell, was preincubated for 1 h at 37°C with different dilutions of anti-gp64 monoclonal antibody and added to monolayers of human dermal fibroblasts. (d) Role of the Ad5GFP moiety in cell internalization of BV^CAR-Ad5GFP complex. Human MSCs were transduced with a mixture of nonpseudotyped, GFP-expressing baculoviral vector BV-GFP and Ad5Luc (Ad5-unbound BV-GFP; leftmost bars) or a mixture of CAR-pseudotyped, GFP-expressing baculoviral vector BV^CARGFP with Ad5Luc (Ad5-bound BV-GFP; rightmost bars). BV-GFP and BV^CARGFP were used at constant MOIs each (MOI of 500), and Ad5Luc was used at different MOIs (0, 50, and 100). The transduction efficiency was expressed as the percentage of GFP-positive cells, assayed by flow cytometry (means of three separate experiments ± SEMs). *, P < 0.05; **, P < 0.01; ns, no significant difference.

FIG. 11.

Role of RGD-dependent integrins in BV^CAR-Ad5GFP-mediated transduction. (a) Efficiency of transduction of permissive cells by Ad5EGD-GFP mutant versus that by Ad5GFP. Aliquots of HEK-293 cells (1.75 × 10⁵/well) were infected for 1 h with Ad5EGD-GFP or control vector Ad5GFP at different MOIs, as indicated on the x axis, and the percentage of GFP-positive cells was determined by flow cytometry at 48 h p.i. (means of three separate experiments ± SEMs). (b) Growth rate of Ad5EGD-GFP mutant versus that of Ad5GFP in permissive cells. Samples of 5 × 10⁴ HEK-293 cells were infected at an MOI of 10 PFU/cell at 37°C for 1 h, rinsed once, and further incubated in culture medium at 37°C. Cells were harvested at 24, 48, and 72 h p.i. and lysed by freeze-thawing in 0.2 ml PBS, and titers of soluble supernatants were determined on HEK-293 cells. Titers were expressed as PFU/cell. (c and d) Efficiency of transduction by the BV^CAR-Ad5EGD-GFP complex. Dermal fibroblasts (DFs) (c) and MSCs (d) were transduced by Ad5EGD-GFP alone or a mixture of BV^CAR at a constant MOI (500 vp/cell) and Ad5EGD-GFP at increasing MOIs, as indicated on the x axis. The transduction efficiency was expressed as the percentage of GFP-positive cells, assayed by flow cytometry at 48 h p.i. (means of three separate experiments ± SEMs). *, P < 0.05; **, P < 0.01.

FIG. 12.

EM analysis of the early steps of virus-cell interaction between BV^CAR-Ad5GFP complex and CAR-negative cells. Monolayers of CHO cells were incubated with BV^CAR-Ad5GFP (complex generated with an Ad5GFP-to-BV^CAR ratio of 1:25) for 1 h at 37°C, and cells were harvested and processed for EM analysis. Panels a to c show the steps of attachment of BV^CAR-Ad5GFP at the plasma membrane and formation of clathrin-coated vesicles (CCV). Panels d and e show coendocytosed virions of BV^CAR and Ad5GFP (Ad) within intracytoplasmic vesicles. In panel g, an adenovirion is seen in the process of vesicular escape. In panel h, two adenovirions are free within the cytoplasm, and electron-dense particles reminiscent of adenoviral cores are seen at the nuclear pore complex (NPC). Panel f shows an intravesicular BV^CAR nucleocapsid released from the baculoviral envelope. N, nucleus; PM, plasma membrane.