Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes

Hendrik J. Rademaker1, Mohamed A. Abou El Hassan1,2, Gijs A. Versteeg1, Martijn J. W. E. Rabelink1 and Rob C. Hoeben1

Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands1
Department of Medical Oncology, Free University Medical Center, Amsterdam, The Netherlands2

Author for correspondence: Rob Hoeben. Fax +31 71 527 6284. e-mail R.C.Hoeben{at}lumc.nl


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Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a {Delta}E1{Delta}E3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing {Delta}E1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.


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Adenoviruses (Ad) are a diverse family of viruses. In humans at least 51 different serotypes have been described, which have been divided into six subgroups (formerly species) based on haemagglutination properties with red blood cells, oncogenic potential and G+C content of their DNA (Horwitz, 1995 ). Replication-defective derivatives of serotypes 5 (Ad5) and 2 (Ad2), generated by deletion of sequences of the viral E1, E2 and E4 regions, have been widely used as gene-transfer vehicles in experimental gene therapy (Benihoud et al., 1999 ). Therapeutic application of recombinant Ad vectors is complicated by the presence of replication-competent adenoviruses (RCA). The presence of RCA in the vector batches may lead to inflammation and tissue damage in the recipient (Fallaux et al., 1999 ; Hermens & Verhaagen, 1997 ). In addition, RCA can mobilize the Ad vector (Imler et al., 1995 ), and this could lead increased vector shedding. The development and implementation of improved helper cells (Fallaux et al., 1998 ) and vectors (Hehir et al., 1996 ), as well as stringent quality-control measures (Roitsch et al., 2001 ; Fallaux et al., 1999 ), can virtually eliminate the problem of RCA in the vector batches.

However, this does not solve the mobilization problem completely. It is conceivable that after receiving gene therapy, patients develop a viraemia with a wild-type (wt) Ad. Indeed, administration of wt Ad5 into lungs of cotton rats previously exposed to replication-defective Ad5 vectors led to mobilization of the vector (Imler et al., 1995 ). The risk of a mobilization by wt Ad of the same serotype is limited, as most patients will develop immunity against the vector serotype (Harvey et al., 1999 ). It is not known, however, whether the vectors can be mobilized by other serotypes, viz. serotypes different from the one from which the vector had been derived. To study this possibility, we determined whether wt Ads of subgroups A, B, C and E could mobilize the vector Ad5CMVluc, a {Delta}E1{Delta}E3-Ad5 vector carrying the firefly luciferase gene as a reporter.

Firstly, we set up a marker-mobilization assay. HepG2 human hepatoma cells were cultured in 24-well plates in DMEM containing 8% FCS. At 90% confluency the cells were infected with an RCA-free stock of Ad5CMVluc at an m.o.i. of 10. Twenty-four hours post-infection (p.i.) the cells were washed with PBS and exposed, at an m.o.i. of 5, to stocks of Ads of subgroups A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) that had been purified twice on CsCl gradients. All Ad titres were determined by plaque assay on 911 cells (Fallaux et al., 1996 ). At this m.o.i. all wt Ads gave complete CPE at 60 to 120 h p.i. (data not shown). As controls, HepG2 cell cultures were infected with the {Delta}E1{Delta}E3-Ad5 vector Ad5CMVlacZ, or mock infected. In a parallel experiment, the assay was performed in the presence of 20 µg/ml cytosine arabinoside (ara-C) in the culture medium. This concentration of ara-C effectively inhibits Ad replication in HepG2 cells (data not shown). Three days after the initial infection, CPE was apparent in all cultures. At that time, the cells were harvested with the medium and the virus was released from the cells by three cycles of freeze–thawing. After the last cycle, the lysates were cleared by centrifugation for 2 min at 1600 g. The amount of mobilized Ad5CMVluc could be determined by measuring the amount of luciferase virus present in the lysate. To quantify the luciferase virus, HepG2 cells were infected with Ad derived from the HepG2 lysates and luciferase activity was determined 18 h p.i. To inhibit replication of the wt Ads present in the lysates, which may affect the luciferase activity, 20 µg/ml ara-C was added to the medium of the indicator cells. The results of the mobilization assays are depicted in Fig. 1(A). Luciferase expression in HepG2 cells infected with virus derived from the Ad5CMVLuc- and Ad5CMVlacZ-coinfected cells was as low as in HepG2 cells infected with virus derived from Ad5CMVLuc and in PBS-treated cells. So, no significant mobilization had occurred either in the mock-infected cells or in the Ad5CMVlacZ-infected cells. In contrast, luciferase activity was readily detectable in all assays where wt Ads had been added, suggesting that all serotypes could mobilize Ad5CMVluc. Similar results were obtained when the cells were harvested 5 days p.i. with Ad5CMVluc (data not shown). The amount of Ad5CMVluc produced upon coinfection by wt Ad5 was only 10-fold lower than the yield of Ad5CMVluc in the E1-complementing cell line 911 (compare the luciferase activity per cell in the wt Ad5 co-infection in Fig. 1A with the levels in 911 cells in Fig. 2A).



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Fig. 1. Luciferase expression in HepG2 cells infected with mobilized Ad5CMVLuc. HepG2 cells were infected with Ad5CMVLuc (m.o.i.=10) and 24 h p.i. were co-infected with wt Ads (m.o.i.=5) in the presence or absence of 20 mg/ml ara-C. As negative controls, Ad5CMVluc-infected cells were co-infected with Ad5CMVLacZ (m.o.i.=5) or mock-infected, either with or without ara-C. Cells were harvested 48 h co-post-infection and freeze–thawed three times to release the virus. Dilutions of the virus were used to infect HepG2 cells. Eighteen hours after infection cells were lysed and assayed for luciferase activity (A). The graph depicts the means (±1 SD) of three experiments. In parallel, low molecular mass DNA was extracted from infected cells according to the Hirt method and digested with PvuI. Ad5CMVLuc DNA was detected by a radiolabelled Luc-specific probe in Southern analysis (B). The arrow indicates the hybridizing 6 kb luciferase-gene fragment.

 


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Fig. 2. Mobilization of rAd5Luc by trans-complementation of Ad12E1. HepG2, 911, HER-RIC-c4, HER-RIC-c7, HER-SV40(T3·2) and HER-SV40(F1·2) cells were exposed to Ad5CMVluc (m.o.i.=10) in the presence or absence of 20 mg/ml ara-C. Forty-eight hours p.i., cells were harvested and the virus was released by three cycles of freeze–thawing. Subsequently, HepG2 cells were exposed to dilutions of the sample to quantify the amount of luciferase virus. Eighteen hours p.i., cells were lysed and assayed for luciferase expression (A). The graph depicts the means (±1 SD) of three experiments. In parallel, low molecular weight DNA was extracted from these cells according to the Hirt method. DNA was digested with PvuI. Ad5CMVLuc DNA was detected by a radiolabelled Luc fragment in Southern analysis (B). The arrow indicates the hybridizing 6 kb luciferase-gene fragment.

 
To confirm replication of Ad5CMVluc DNA after infection of the cells with wt Ads, low-molecular mass DNA was extracted by Hirt isolation (Hirt, 1967 ) from HepG2/Ad5CMVluc cells infected with the various wt Ads. Southern analysis of PvuI-digested Hirt DNA with a luciferase gene-specific probe demonstrated replication of the Ad5CMVluc vector (Fig. 1B). Replication (Fig. 1B), as well as the mobilization (Fig. 1A), was sensitive to 20 µg/ml ara-C, indicating that replication is essential for mobilization of the vector. The data suggest that all Ad serotypes tested can mobilize the Ad5CMVluc vector.

Two mechanisms could contribute to the replication and mobilization of the Ad5CMVluc vector. Firstly, the E1 region of the wt virus could functionally complement the E1 deletion of the vector, and activate expression of the Ad5 genes of the vector. In the ‘E1 complementation’ model the vector would be replicated by and packaged in its homologous proteins. Thus, the presence of the E1 proteins of the wt Ad would be sufficient to mobilize the vector. Alternatively, in the ‘facilitated replication and packaging’ model, the wt Ad’s E1 region alone is unable to activate expression of the vector genome. Hence, mobilization can only occur if the wt Ad-derived proteins other than, or in addition to, E1 facilitate replication and packaging of the vector DNA.

To test whether expression of E1 is sufficient to replicate the Ad5CMVluc vector we used a panel of human embryonic retinoblasts (HER) cell lines transformed with the E1 region of Ad12 and, as controls, cells transformed with the SV40 early region (van den Heuvel et al., 1992 ). As negative and positive controls we used cell lines HepG2 and 911 (Fallaux et al., 1996 ), an HER line transformed with the E1 region of Ad5, respectively. These cell lines were infected with the Ad5CMVluc at an m.o.i. of 10. At 3 days p.i., the cells and media were harvested, freeze–thawed (three cycles), and assayed for the presence of the Ad5CMVluc vector by infecting HepG2 cells with Ad derived from the HepG2 lysates followed by luciferase assay (Fig. 2A). Ad5CMVluc propagation was detectable neither in the HepG2 cells nor in the SV40-transformed HER-SV40(T3.2) and HER-SV40(F1.2) cells. In contrast, the HER-RIC-c4 and HER-RIC-c7 cells produced readily detectable amounts of reporter virus. The amplification factor, that is the ratio of the Ad5CMVluc production in the cultures divided by the Ad5CMVluc production in the ara-C-treated cultures, was only moderately lower than the amplification factor obtained in the Ad5E1-transformed 911 cells. Virus replication was sensitive to ara-C. Similar results were obtained if the viruses were harvested 5 days p.i. (data not shown). Southern analysis confirmed replication of the vector in the Ad5E1- and Ad12E1-transformed HER lines, and not in the SV40-transformed HER lines (Fig. 2B). Taken together, these data show that wt Ads of subgroups A, B, C and E can mobilize {Delta}E1{Delta}E3-Ad5 vectors and that for Ad12 at least, trans-complementation by E1 is sufficient for mobilization at least for Ad12E1.

Our data confirm and extend the earlier observation that BamHI-digested wt Ad12 (subgroup A) and Ad7 (subgroup B) could efficiently complement an E1 deletion of Ad2 (subgroup C) (Brusca & Chinnadurai, 1981 ). Also, hybrid Ad5 and Ad12 viruses in which the E1 region had been replaced (in part) by the corresponding region of Ad12 and Ad5, respectively, were demonstrated to be viable (Bernards et al., 1983 ). However, another study showed that a defect in the E1 region of Ad2 could only be complemented poorly by Ad9 (subgroup D) (Jannun & Chinnadurai, 1987 ). In addition, we show here that Ad12E1-transformed HER cell lines complement the E1 deletion in Ad5 and allow the Ad5 vector to be propagated.

It remains to be established if the ‘facilitated replication and packaging’ model really contributes to the mobilization of {Delta}E1{Delta}E3-Ad5 vectors. It has been shown that Ad5-derived helper-dependent Ad vectors can be amplified and packaged into Ad2-derived capsids (Morral et al., 1999 ), demonstrating the feasibility of the model. It should be borne in mind, however, that that Ad2 and Ad5 are highly similar ‘twin’ serotypes and have origins of DNA replication and packaging signals that are identical in sequence. Indeed, it has been shown recently that Ad12 (subgroup A), Ad7 (subgroup B) and Ad 17 (subgroup D) cannot complement the defect of Ad5 mutant pm8001 (Zhang et al., 2001 ). This mutant cannot package its DNA due to a mutation in the L1 52/55 kDa gene. This indicates that the DNA packaging system of different serotypes cannot productively interact with Ad5 DNA. Thus, mobilization by E1 complementation appears to be the more general mechanism.

Our data demonstrate that Ad5-derived vectors can be mobilized not only by wt viruses of the homologous serotype, but also by different serotypes. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication the genome packaging of DNA (Imler et al., 1995 ) are further restricted.


   Acknowledgments
 
This work was supported in part by the Technology Foundation STW (Program LGN 66.3977).


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Received 5 December 2001; accepted 24 January 2002.