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Journal of Virology, October 1999, p. 8689-8695, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Chimeric Type 2 Adenovirus Vector with a Type 17 Fiber Enhances Gene Transfer to Human Airway Epithelia

Joseph Zabner,1,* Miguel Chillon,1 Teresa Grunst,1 Thomas O. Moninger,1 Beverly L. Davidson,1 Richard Gregory,2 and Donna Armentano2

Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242,1 and Department of Gene Therapy, Genzyme Corp., Framingham Massachusetts 017092

Received 16 December 1998/Accepted 21 June 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In studies of the genetic disease cystic fibrosis, recombinant adenovirus type 2 (Ad2) and Ad5 are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia. However, earlier work has shown that human airway epithelia are resistant to infection by Ad2 and Ad5. Therefore, we examined the efficiency of other adenovirus serotypes at infecting airway epithelia. We found that several serotypes of adenoviruses, in particular, wild-type Ad17, infected a greater number of cells than wild-type Ad2. The increased efficiency of wild-type Ad17 could be explained by increased fiber-dependent binding to the epithelia. Therefore, we constructed a chimeric virus, Ad2(17f)/beta Gal-2, which is identical to Ad2/beta Gal-2 with the exception of having the fiber protein of Ad17 replace Ad2 fiber. This vector retained the increased binding and efficiency of gene transfer to well-differentiated human airway epithelia. These data suggest that inclusion of Ad17 fiber into adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Recombinant adenoviruses are used as vectors for gene transfer to a wide variety of cells and tissues, and it is hoped that they may prove useful for gene therapy. In the genetic disease cystic fibrosis, recombinant adenoviruses are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia.

The first steps in adenovirus infection are thought to involve primarily two proteins in the capsid: fiber and penton base (12, 15, 16). The fiber protein is important for binding to a high-affinity fiber receptor. In immortalized cell lines (KB, A459, and HeLa) this receptor is thought to be present in the range of 3,000 to 10,000 receptors/cell (6, 27, 28). Cells that lack fiber receptor activity are poorly infected by adenovirus (34). Recent work identified the coxsackie B virus and adenovirus type 2 (Ad2) and Ad5 receptor (CAR), which binds adenovirus fiber and may be involved in the pathogenesis of adenovirus infection (4, 26). The major histocompatibility complex (MHC) class I alpha -2 domain also mediates adenovirus binding and infection (20). After binding to the fiber receptor, penton base interaction with alpha Vbeta integrins facilitates internalization via receptor-mediated endocytosis (16, 25, 38). This penton base-alpha vbeta interaction appears to be less important than the fiber-fiber receptor interaction. Neither mutations in the RGD motif of penton base nor competition by penton base protein completely prevents infection of cells by adenovirus (16, 18, 25, 38).

Most of the studies on the mechanism of adenovirus infection done with cell lines have concluded that adenovirus infection and adenovirus-mediated gene transfer are very efficient. However, the efficiency of Ad2 and Ad5-mediated gene transfer to the human airway epithelia both in vitro and in vivo is quite limited (1, 13, 14, 17, 29, 40, 43). The inefficiency appears to be due to the lack of fiber-mediated binding to the apical surface of the airway epithelia (43). Consistent with this, human airway epithelia do not express human CAR (hCAR) on the apical surface (30, 36). Furthermore, MHC class I is absent in the apical surface, and both receptors are polarized to the basal surface, where they are physically unable to bind the adenovirus fiber protein (36).

Recent data suggest that interventions which increase virus binding increase adenovirus-mediated gene transfer (9, 10). These studies suggest that binding is the rate-limiting step for adenovirus-mediated gene transfer to the airway epithelia. In this study, we examined the possibility that the fiber protein from other adenovirus serotypes might be more efficient in binding and mediating infection of human airway epithelia.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Human airway epithelia. Airway epithelial cells were obtained from surgical polypectomies of non-cystic fibrosis patients or from trachea and bronchi of lungs removed for organ donation. Cells were isolated by enzyme digestion as previously described (32). Freshly isolated cells were seeded at a density of 5 × 105 cells/cm2 onto collagen-coated, 0.6-cm-surface area Millicell polycarbonate filters (Millipore Corp., Bedford, Mass.). The cells were maintained at 37°C in a humidified atmosphere of 7% CO2 and air. Twenty-four hours after plating, the mucosal medium was removed and the cells were allowed to grow at the air-liquid interface (21, 39). The culture medium consisted of Dulbecco modified Eagle medium-Ham's F12 (1:1), 5% Ultroser G (Biosepra SA, Cedex, France), 100 U of penicillin per ml, 100 µg of streptomycin per ml, 1% nonessential amino acids, and 0.12 U of insulin per ml. Epithelia were tested for transepithelial resistance and for morphology by scanning electron microscopy.

Recombinant adenoviruses. A recombinant adenovirus vector expressing beta -galactosidase, Ad2/beta Gal-2 was prepared as described previously (37) by the University of Iowa Gene Transfer Vector Core at titers of ~1010 infectious units (IU)/ml. Recombinant fiber knob 2, fiber knob 17, and penton base protein were prepared as previously described (3). Wild-type human adenoviruses were purchased from the American Type Culture Collection (Rockville, Md.). To determine the viral particle concentration, the virus was diluted in 10 mM Tris (pH 8.0)-1 mM EDTA-0.1% sodium dodecyl sulfate, and the absorbance at 260 nm was measured. Under these conditions, corrected for dilution, an absorbance of 1 corresponds to 1012 particles/ml. To determine the number of IU, the virus titers were determined by serial dilution assay on 293 cells in quadruplicate as described below.

Construction of the chimeric virus Ad2(17f)/beta Gal-2. A chimeric Ad2/beta Gal-2/fiber Ad17 virus, Ad2(17f)/beta Gal-2, was constructed as follows. PAdORF6 (2) was cut with NdeI and BamHI to remove Ad2 fiber-coding and polyadenylation signal sequences. An NdeI-BamHI fragment containing Ad17 fiber-coding sequence was generated by PCR and ligated along with a simian virus 40 polyadenylation signal into NdeI-BamHI-cut pAdORF6 to generate pAdORF6fiber17. This plasmid was cut with PacI and ligated to PacI-cut Ad2/beta gal-2 genomic DNA. The ligation product was transfected into 293 cells, plaques were picked, and virus was expanded and analyzed by restriction endonuclease digestion. The resulting virus contains the N-terminal 16 amino acids of the tail region from Ad2 and the remainder of the tail, shaft, and knob from Ad17. The yield of Ad2(17f)/beta Gal-2 production was between 3.4 × 102 and 3.4 × 103 particles/cell. To label the chimeric virus proteins, 293 cells were infected with either Ad2 or Ad2(17f)/beta Gal-2 at a multiplicity of infection (MOI) of 20. Thirty-eight hours later the cells were metabolically labeled with [35S]methionine as previously described (32). Cell lysates were electrophoresed on a sodium dodecyl sulfate-10% polyacrylamide gel, which was then dried and autoradiographed.

Viral infection and binding assays. Airway epithelia were allowed to reach confluence and develop a transepithelial electrical resistance, indicating the development of tight junctions and an intact barrier. All epithelia had transepithelial electrical resistance values of >500 Omega  · cm2. Epithelia were allowed to differentiate by culturing for at least 14 days after seeding. Recombinant viruses at an MOI of 50 (in phosphate-buffered saline [PBS]) or the wild-type adenoviruses at an MOI of 10 were added to the apical surface. Following the indicated incubation time, the viral suspension was removed and the epithelia were rinsed twice with PBS. After infection, the epithelia were incubated at 37°C for an additional 30 to 72 h.

To assess binding to airway epithelia, the epithelia were incubated for 5 and 30 min at 4°C with 2,500 particles of [35S]methionine-labeled adenoviruses per cell as previously described (40). Cell-associated 35S-adenovirus was evaluated in sextuplicate by liquid scintillation counting (LKB Wallace, Gaithersburg, Md.) by calculating the percentage of bound counts per minute.

Measurement of beta -galactosidase activity. We measured total beta -galactosidase activity by a commercially available method (Galacto-Light; Tropix, Inc., Bedford, Mass.). Briefly, after being rinsed with PBS, cells were removed from filters by incubation with 120 µl of lysis buffer (25 mM Tris-phosphate [pH 7.8], 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100) for 15 min. Light emission was quantified in a luminometer (Analytical Luminescence Laboratory, San Diego, Calif.).

Determination of wild-type adenovirus infection. We estimated the number of cells infected with the different serotypes of wild-type adenovirus by antihexon staining (42). HeLa cells and airway epithelia cultured on permeable filter supports were studied at 30 h after infection. Cells were fixed with acetone-methanol and stained with a polyclonal fluorescein isothiocyanate (FITC)-labeled anti-hexon antibody (Chemicon, Temecula, Calif.). Hexon-positive cells were counted by fluorescence microscopy. This method allows detection of infected cells by staining for the most abundant adenovirus protein, hexon. The antibody recognized hexon protein from all adenovirus serotypes.

hCAR cDNA electroporation of CHO cells. The pcDNA3-hCAR plasmid was cloned from pcDNA1-hCAR (a generous gift from J. M. Bergelson) by restricting hCAR from pcDNA1 into pcDNA3 with BamHI and NotI. Chinese hamster ovary (CHO) cells (107) were electroporated with 20 µg of plasmid pcDNA3-hCAR, pcDNA3GFP, or pCMV-CFTR. The cells were plated on 24-well plates at 250,000 cells/well and grown overnight at 37°C.

Transmission electron microscopy. Ad2/beta Gal-2 and Ad2(17f)/beta Gal-2 were processed for transmission electron microscopy by using a negative stain technique. Fifteen-microliter drops of freshly thawed viral suspensions were placed on glow-discharged collodion-carbon-coated 400-mesh copper grids and left for 3 min. The solution was wicked off with filter paper and replaced with 1% aqueous uranyl acetate for 30 s. After removal of this solution, grids were allowed to dry and imaged in a Hitachi H-7000 transmission electron microscope.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Wild-type adenovirus infection of ciliated airway epithelia. We have previously shown that after 2 weeks in culture, primary cultures of human airway epithelial cells grown at the air-liquid interface differentiate into an epithelium covered with a mat of cilia. Furthermore, these epithelia are resistant to Ad2 infection and lack detectable levels of CAR and MHC class I at the apical surface (30, 36). We investigated the relative infection efficiencies of different adenovirus serotypes from the six subgroups (A to F) on well-differentiated ciliated human airway epithelia. Epithelia were infected, with a short incubation time (30 min), with wild-type Ad2, Ad3, Ad4, Ad7, Ad9, Ad14, Ad17, Ad19, Ad26, Ad30, Ad31, and Ad41 at an MOI of 10. The epithelia were studied 30 h after infection by labeling infected cells with FITC-conjugated antihexon antibody, as previously described (40). Figure 1 (top) shows examples from airway epithelia infected with different serotypes. We found that several serotypes of adenoviruses infected a greater number of cells than wild-type Ad2 (Fig. 1, bottom). In particular, wild-type Ad17, Ad9, and Ad4 (subgenera DII, DI, and E, respectively) infected 43, 17, and 12 times more cells, respectively, than wild-type Ad2 (subgenus C). However, at this point it was not clear if the apparent differences in infection were due to different binding affinities of fiber, binding mediated through different receptors, or other factors such as binding via other capsid proteins. In addition, because hexon production depends on virus replication, we could not exclude the possibility that the differences in apparent infection were due to variations in replication time for the different serotypes.



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  		FIG. 1.   Expression of hexon protein by well-differentiated human airway epithelia infected by different adenovirus serotypes (serotypes 2, 3, 4, 7, 9, 14, 17, 19, 26, 30, 31, and 41). (Top) Immunocytochemistry of airway epithelia 30 h following application to the apical surface of 12 different adenovirus serotypes at an MOI of 10 for 30 min. (Bottom) Number of cells infected by different adenovirus serotypes. Data are the mean numbers (± standard errors of the means) of cells expressing hexon protein per low-power field (n = 3).

Effect of incubation time on infection of ciliated airway epithelia by wild-type adenoviruses. We had initially noticed that a prolonged incubation time would allow Ad2 to infect ciliated airway epithelia by a fiber-independent mechanism (43). We compared the effects of short (30-min) and prolonged (12-h) incubation times on infection of ciliated airway epithelia by Ad17. Figure 2 shows that as previously reported (40, 43), a short incubation time resulted in a decrease in the number of wild-type-Ad2-infected cells compared to that with a long incubation time (8.3 ± 1.2 versus 142.3 ± 48 hexon-positive cells/field). In contrast, wild-type Ad17 infected similar numbers of cells when applied for a short or long incubation time (222.3 ± 12 versus 296 ± 26 hexon-positive cells/field). These data suggest that the advantage of Ad17 over Ad2 is related to the kinetics of binding and infection.


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  		FIG. 2.   Effect of incubation time on infection of well-differentiated human airway epithelia. The apical surface of airway epithelia was exposed to wild-type Ad17 or wild-type Ad2 at an MOI of 50 for either 30 min or 12 h. Data are photomicrographs of the immunocytochemistry of airway epithelia stained with an FITC-labeled antihexon antibody 30 h later.

Ad17 shows greater binding to ciliated airway epithelia than Ad2. To determine if the improved infection efficiency of Ad17 relied on increased binding to well-differentiated airway epithelia, we performed binding assays. Epithelia were incubated for either 5 or 30 min at 4°C with [35S]methionine-labeled wild-type Ad17 or wild-type Ad2. Cell-associated 35S-adenovirus was evaluated by liquid scintillation counting. Figure 3 shows that differentiated airway epithelia bound wild-type Ad17 better than wild-type Ad2. These data may in part explain the advantage of wild-type Ad17 over wild-type Ad2.


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  		FIG. 3.   Binding of [35S]methionine-labeled wild-type Ad17 and wild-type Ad2 to airway epithelia. The figure shows the percentage of Ad2 and Ad17 binding by well-differentiated airway epithelia at a particle/cell ratio of 2,500. The left panel shows the results of a 5-min incubation, and the right panel shows the results of a 30-min incubation. Bars show means (± standard errors of the means) of the percentage of total counts per minute added that remained epithelium associated after a PBS rinse. Asterisks indicate significance (P < 0.05).

A recombinant Ad2 expressing Ad17 fiber retains the increased binding of Ad17. Fiber protein from wild-type Ad2 differs from that of wild-type Ad17. The amino acid sequence of the Ad17 fiber shaft is shorter than that of the Ad2 fiber, suggesting a shorter fiber. Moreover, Ad17 and Ad2 have only 40% homology in the region of the fiber knob which binds to the fiber receptor (24). Therefore, we speculated that the binding advantage of Ad17 was due to a different interaction between fiber 17 and its receptor.

To study the relative importance of fiber 17, we therefore constructed an E1-negative, E4-negative, open reading frame 6-positive Ad2 containing the Escherichia coli beta -galactosidase gene under control of the cytomegalovirus promoter. This virus, Ad2(17f)/beta Gal-2, is identical to Ad2/beta Gal-2 with the exception of the fiber protein. The type 2 fiber gene in this virus encodes the N-terminal 16 amino acids of the tail region from Ad2 and the remainder of the tail, shaft, and knob from Ad17. Figure 4A shows that the chimeric adenovirus had a fiber knob that was 0.41 the length of the Ad2/beta Gal-2 fiber. To further characterize the chimeric structure of Ad2(17f)/beta Gal-2, we metabolically labeled with [35S]methionine 293 cells infected with either Ad2 or Ad2(17f)/beta Gal-2. Figure 4B shows that Ad2/beta Gal-2 virus contains an expected ~40-kDa band for fiber protein 17 and lacks the expected 72-kDa band for Ad2 fiber. These data confirm the chimeric structure of Ad2(17f)/beta Gal-2.



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  		FIG. 4.   Chimeric recombinant Ad2 fiber protein from Ad17. (A) Transmission electron photomicrographs of Ad2/beta Gal-2 and Ad2(17f)/beta Gal-2. Arrows indicate examples of the fiber proteins of each virus. Bar, 100 nm. (B) Fiber protein synthesis by Ad2 and Ad2(17f)/beta Gal-2. 293 cells were infected with Ad2 or Ad2(17f)/beta Gal-2 and metabolically labeled with [35S]methionine. The SDS-polyacrylamide gel shows the appearance of a new ~40-kDa band that corresponds to fiber 17 and the disappearance of the fiber 2 band on the cell lysate infected with Ad2(17f)/beta Gal-2.

To compare the infection efficiency of Ad2/beta Gal-2 with that of the Ad2(17f)/beta Gal-2 chimera, we needed to apply the same number of IU of both vectors. We initially found that the d2(17f)/beta Gal-2 virus infected 293 cells poorly. This observation posed the question of whether differences in infection by the two vectors could be due to an error in estimating the number of IU on 293 cells. Therefore, we used prolonged incubation (43) and adenovirus CaPi coprecipitates (10) to develop a titer assay that bypassed the relative inefficiency of infection of individual viruses. As shown in Fig. 5, we found that the resistance of 293 cells to infection by Ad2(17f)/beta Gal-2 could be overcome either by increasing the incubation time with the virus or by using CaPi coprecipitates which allow infection without the requirements for a fiber receptor (10, 43). For the studies outlined below we matched preparations of adenovirus with similar particle/IU ratios (long incubation time) for comparisons between the vectors Ad2/beta Gal-2 (particle/IU ratio of 81) and Ad2(17f)/beta Gal-2 (particle/IU ratio of 85). Therefore, both the MOIs and the particle numbers were similar for the two viruses.


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  		FIG. 5.   Effect of incubation time and adenovirus-CaPi coprecipitates on the determination of the number of IU on 293 cells. 293 cells were seeded on 96-well plates and grown until they were 70% confluent. Serial dilutions of 293 medium (140 µl) containing 1010 particles of virus were added to the 293 cells. After 30 min or 72 h, the virus was aspirated and washed twice. For the CaPi coprecipitates, CaCl was added to a suspension of 1010 particles in Eagle minimal essential medium to a final concentration of 5.6 mM. After 30 min, the adenovirus-CaPi coprecipitates were aspirated. Seventy-two hours later, the cells were fixed and stained with an antihexon FITC-labeled antibody. Data are the ratio of adenovirus particles to infectious units determined by each assay (n = 4).

To test if the Ad2(17f)/beta Gal-2 virus bound to ciliated airway epithelia via a fiber 17-mediated mechanism, we measured binding of [35S]methionine-labeled Ad2(17f)/beta Gal-2 to differentiated airway epithelia following a 30-min incubation. As shown in Fig. 6, we found that well-differentiated airway epithelia bound Ad2(17f)/beta Gal-2 in a range similar to that of wild-type Ad17 (Fig. 3) and that binding was greater than that observed earlier with wild-type Ad2 (Fig. 3) and recombinant Ad2/beta Gal-2 (40). To test if the increased binding was due to the fiber protein-receptor interaction, we tested the effect of excess fiber protein on Ad2(17f)/beta Gal-2 binding to airway epithelia. We found that addition of type 17 fiber knob protein blocked binding of Ad2(17f)/beta Gal-2 to airway epithelia, whereas type 2 fiber knob did not. Thus, the improved binding of Ad2(17f)/beta Gal-2 to airway epithelia can be explained by the different fiber protein.


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  		FIG. 6.   Effect of fiber knob 2 and fiber knob 17 on [35S]methionine-labeled Ad2(17f)/beta Gal-2 binding. The percentage of Ad2(17f)/beta Gal-2 binding by well-differentiated airway epithelia at a particle/cell ratio of 2,500 and in the presence of fiber knob 2 and fiber knob 17 is shown. Bars show means (± standard errors of the means) of the percentage of total counts per minute added that remained epithelium associated after a PBS rinse (n = 6). Asterisks indicate significance (P < 0.05).

Ad2(17f)/beta Gal-2 mediates gene transfer to airway epithelia more efficiently than Ad2/beta Gal-2. We tested the ability of Ad2(17f)/beta Gal-2 to express beta -galactosidase in differentiated airway epithelia. The Ad2(17f)/beta Gal-2 chimera at an MOI of 50 generated between 15 and 95 times more beta -galactosidase activity than Ad2/beta Gal-2 at a similar IU-to-particle ratio of 85. Figure 7 shows average results from four different experiments. These data confirmed the hypothesis that a recombinant adenovirus expressing fiber protein from wild-type Ad17 improves the efficiency of gene transfer to ciliated airway epithelia.


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  		FIG. 7.   Chimeric Ad2(17f)/beta Gal-2-mediated beta -galactosidase expression in human airway epithelia. Well-differentiated airway epithelia were exposed to either Ad2/beta Gal-2 or Ad2(17f)/beta Gal-2 at an MOI of 50 for 30 min. The vector was then removed, and the epithelia were cultured for an additional 2 days before analysis. Data are log means (± standard errors of the means) of beta -galactosidase activity (n = 6). Asterisks indicate significance (P < 0.01). L. U., light units.

Is Ad2(17f)/beta Gal-2 infection of airway epithelia dependent on Ad17 fiber or on penton base? Roelvink et al. (33) showed that a chimeric Ad5 expressing the fiber protein from Ad9 resulted in an increased penton base-mediated binding and infection of cells that lack fiber receptor. They speculated that a shorter fiber protein made the penton base more accessible to interact with alpha vbeta integrins. Because well-differentiated airway epithelia do not express alpha vbeta integrins on the apical surface (13, 14), it is unlikely that a similar mechanism could explain the advantage of Ad2(17f)/beta Gal-2 in airway epithelia. Nevertheless, we investigated the ability of Ad2(17f)/beta Gal-2 to mediate gene transfer to human airway epithelia by fiber-dependent and by penton-base dependent mechanisms. Figure 8 shows that Ad2(17f)/beta Gal-2-mediated beta -galactosidase expression was partially blocked by 70 µg of Ad17 fiber knob protein per ml. In contrast, neither Ad2 fiber knob protein nor Ad2 penton base protein blocked Ad2(17f)/beta Gal-2. The data suggest that Ad2(17f)/beta Gal-2 binds and infects human ciliated airway epithelia via a fiber 17 receptor-mediated mechanism that is independent of the penton base.


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  		FIG. 8.   Effect of fiber knob and penton base on Ad2(17f)/beta Gal-2-mediated gene transfer to human airway epithelia. Type 2 fiber knob, type 17 fiber knob, and penton base protein (70 µg/ml) or solution alone were applied to human airway epithelia. Ad2(17f)/beta Gal-2 at an MOI of 50 was then added and left for 30 min at 4°C. beta -Galactosidase was measured 48 h later. Data are means ± standard errors of the means (n = 9 in each group). Asterisks indicate significance (P < 0.02). L. U., light units.

Interaction of Ad17 fiber with hCAR. Human airway epithelia express the Ad2 and Ad5 fiber receptors hCAR and MHC class I (30, 36). However, neither receptor is present in the apical membranes of well-differentiated airway epithelia at a level that can be detected by immunocytochemistry or at a level that allows fiber-dependent infection by Ad2. Ad9, a member of the same subgenus as Ad17, has also been shown to interact with hCAR (33).

We therefore investigated the ability of the chimeric adenovirus to infect CHO cells that transiently expressed hCAR cDNA. We electroporated CHO cells with a plasmid that expresses hCAR (pcDNA3hCAR), a plasmid that expresses green fluorescence protein (pcDNA3GFP), or a plasmid that expresses CFTR (pCMV-CFTR) as a control. Over 50% of CHO cells electroporated with pcDNA3GFP expressed green fluorescence protein by fluorescence-activated cell sorting (data not shown). Consistent with the report of Bergelson et al. (4), Ad2/beta Gal-2-infected CHO cells expressed hCAR, but control CHO cells did not (Fig. 9A). Furthermore, infection could be competed with fiber knob 2 protein. Interestingly, fiber knob 17 protein also inhibited infection by Ad2/beta Gal-2, suggesting that the Ad17 fiber may interact with hCAR.


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  		FIG. 9.   Effect of recombinant hCAR expression on Ad2/beta Gal-2- and Ad2(17f)/beta Gal-2-mediated gene transfer in CHO cells. CHO cells were electroporated with plasmid pcDNA3hCAR, pcDNA3GFP, or with pCMV-CFTR as control. (A) Data are the beta -galactosidase activity of hCAR-expressing CHO cells infected with Ad2/beta Gal-2 at an MOI of 50. Both type 2 and type 17 fiber knob (both at 70 µg/ml) block the infection. (B) Data are the beta -galactosidase activity of hCAR-expressing CHO cells infected with Ad2(17f)/beta Gal-2 at an MOI of 50. Only type 17 fiber knob (70 µg/ml) blocked the infection of the chimeric virus. Data are means ± standard errors of the means (n = 12 in each group). Asterisks indicate significance (P < 0.01). L. U., light units.

Similar to the case for Ad2 and Ad5 infection, CHO cells were resistant to infection by Ad2(17f)/beta Gal-2 (Fig. 9B). However, transient expression of hCAR cDNA increased gene transfer by the chimeric adenovirus. Figure 9B also shows that Ad17 fiber knob protein blocked infection of Ad2(17f)/beta Gal-2, whereas the Ad2 fiber knob protein did not. These data suggest that the receptor required for fiber-dependent infection with Ad2 (hCAR) can also mediate infection by Ad17. The finding that Ad2 fiber knob failed to compete infection of Ad2(17f)/beta Gal-2 suggests either that the affinity of Ad17 fiber knob for hCAR is higher than the affinity of Ad2 fiber knob or that Ad17 fiber may bind to a different epitope of hCAR as well as interfere with type 2 fiber binding. Finally, although Ad17 fiber binds to hCAR, we cannot exclude the possibility that Ad17 fiber may bind to additional receptors expressed on the apical surface of airway epithelia.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Although there is little doubt that adenovirus vectors can deliver genes to airway epithelia, both in vitro and in vivo, the efficiency of gene transfer is much less than desired. Lack of fiber receptor at the apical membrane appears to be the rate-limiting step explaining the inefficiency of subgroup C adenoviruses (14, 17, 19, 41, 43). At present it is difficult to conceive of a way to increase hCAR or MHC class I expression on the apical surface of airway cells. However, alternatives that increase nonspecific binding to airway epithelia by covering the virus with cationic substances or CaPi coprecipitates have resulted in increased efficiency of gene transfer (9, 10). Another approach, yet to be tested with airway epithelia, relies on changes in the capsid proteins that lead to binding to a different receptor (8, 11, 23, 33, 35). However, what receptor to target on airway epithelia is still not known.

In this work we used a different approach, searching for a serotype of adenovirus that might be able to use a different receptor present on the apical surface of airway epithelia. Using a similar approach, Croyle et al. (5) determined that the binding of wild-type Ad41 to intestine is better than that of Ad5. Our data show that wild-type Ad17 can bind and infect differentiated human airway epithelia more efficiently than wild-type Ad2. Furthermore, a chimeric Ad2 expressing type 17 fiber retained the advantages in binding, infection, and gene transfer. We have also recently reported that Ad17 and the chimeric Ad2 virus expressing Ad17 fiber were more efficient in infecting primary cultures of neurons (4a).

Fiber is a trimeric protein that protrudes from the 12 vertices of the viral capsid. Fiber protein consists of a short conserved N-terminal tail that binds to penton base, a variable-length shaft, and a globular fiber knob, which is responsible for the interaction with the virus receptor at the cell membrane. It has been shown that the cellular tropism of adenoviruses depends on the fiber knob (7, 11, 22, 33). Roelvink et al. (33) recently suggested that two different serotypes of adenovirus (Ad2 and Ad9) might share the same fiber receptor. They found that unlike that of Ad2, Ad9 binding to some cells could not be competed off by excess fiber knob. Because this fiber-independent binding was blocked by a monoclonal antibody to an alpha v integrin, they concluded that the shorter length of fiber 9 (11 nm) relative to fiber 2 (37 nm) permitted fiber-independent binding of Ad9 penton base to alpha v integrins. However, it is unlikely that penton base binding to alpha v integrins is responsible for the increased efficiency of Ad17, because alpha v integrins are not expressed on the surface of airway epithelia (13, 14). Furthermore, in contrast to the results of Roelvink et al. (33), our data show that excess penton base protein failed to block infection of human airway epithelia by Ad2(17f)/beta Gal-2. While Ad17 and Ad9 belong to the same subgenus, they have only 78% homology in the fiber knob sequence (31), and they differ significantly in their abilities to bind and agglutinate human erythrocytes (31). Future studies that investigate mutations that shorten Ad2 fiber, and that compare Ad9 and Ad17 infection on airway epithelia, may yield new insight into the apparent effects of length versus knob sequence on infection.

Why is Ad17 more efficient at binding differentiated airway epithelia than Ad2? Our data show that the affinity of fiber 17 for hCAR is greater than that of Ad2. Moreover, the sequences involved in the increased binding to hCAR are in the fiber knob. However, ciliated airway epithelia do not express hCAR or MHC class I at the apical surface at detectable levels (36). There are at least two possibilities to explain the data. First, in contrast to results obtained with Ad2, very low levels of hCAR at the apical membrane of airway epithelia might be sufficient to mediate Ad17 fiber-dependent binding. This could be due to the higher affinity of Ad17 fiber. Second, a different receptor specific for Ad17 fiber might be expressed at the apical surface of airway epithelia. In either case, the increased transduction observed with the chimeric virus suggests that inclusion of Ad17 fiber or perhaps Ad17 fiber knob in adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.


    ACKNOWLEDGMENTS

We thank Pary Weber, Phil Karp, Janice Launspach, Theresa Mayhew, and Christine McLennan for excellent assistance. We especially appreciate the help of ISOPO and IIAM for the human lungs.

We appreciate the support of the University of Iowa Gene Transfer Vector Core (supported in part by the Roy J. Carver Charitable Trust). This work was supported by the National Heart Lung and Blood Institute, the National Institute of Allergy and Infectious Disease, the Cystic Fibrosis Foundation, and the Roy J. Carver Charitable Trust. J.Z. is a Fellow of the Roy J. Carver Charitable Trust.


    FOOTNOTES

* Corresponding author. Mailing address: University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Phone: (319) 353-5511. Fax: (319) 353-5572. E-mail: Joseph-Zabner{at}uiowa.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, October 1999, p. 8689-8695, Vol. 73, No. 10
0022-538X/99/$04.00+0
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