Department of Virology, Ume University, SE-901 85 Ume
, Sweden1
Author for correspondence: Johan Skog. Fax +46 90 129905. e-mail johan.skog{at}climi.umu.se
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Abstract |
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Introduction |
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An adenovirus fibre protrudes from each of the 12 vertices of the icosahedral adenovirus particle. Gene delivery to cells by adenovirus is initiated by the binding of the adenovirus fibre knob to a cellular receptor, followed by an interaction between the penton base RGD motif and v-integrins in the cell membrane (Wickham et al., 1993
). The coxsackievirus and adenovirus receptor (CAR) can act as a primary receptor for adenoviruses in species A and CF (Roelvink et al., 1998
), although some serotypes in these species seem to use receptors other than CAR (Arnberg et al., 2000a
). Furthermore, adenoviruses from species B use a receptor that is different from CAR (Roelvink et al., 1998
). The
2 domain of major histocompatibility complex class 1 (MHC-1) molecules and the vascular cell adhesion molecule-1 have been described, in addition to CAR, to be receptors for Ad5 (Hong et al., 1997
). The main CAR-binding epitopes of the fibre knob are located in the AB-loop (comprising over 50% of the interfacial proteinprotein interactions) but also additional residues in the B
-sheet, the DE-loop and the FG-loop are also important. Some of these residues are conserved among the CAR-binding adenoviruses (Bewley et al., 1999
; Roelvink et al., 1999
).
Gene transfer to neural cells has gained considerable interest in the field of neuroscience (Hermens & Verhaagen, 1998 ; Kaplitt & Makimura, 1997
). Applications for gene transfer range from treatment of genetic diseases, tumours and acquired degenerative encephalopathies, such as Alzheimers disease and Parkinsons disease, to being a powerful tool in the study of biological mechanisms (Alton & Kitson, 2000
; Choi-Lundberg & Rosenthal, 1999
; Lang et al., 1999
; Miller et al., 1998
; Russell & Hirata, 1998
; Wood et al., 1996
). The in vivo delivery of adenovirus into the brain can induce an immune response regardless of the more protective environment in the CNS. Adenovirus vectors with a deleted early E1 region induce a systemic humoral response in a dose-dependent manner (Kajiwara et al., 2000
). In non-immunized individuals, the cell-mediated immune response against the vector is not sufficient to quench vector-induced gene expression, which can be maintained for months (Thomas et al., 2000
). A subsequent peripheral exposure to the vector will lead to a loss of vector-mediated gene expression by a massive T-lymphocyte infiltration of the brain parenchyma and macrophage/microglial activation targeting the vector-infected cells (Byrnes et al., 1995
, 1996
). It is therefore desirable to improve the efficiency of transduction in order to minimize the dose of virus required. Another strategy to avoid the immune response is to delete all virus-related genes in the vector to avoid leakage of viral proteins from the infected cells (Fisher et al., 1996
; Kochanek et al., 1996
). These high-capacity adenovirus vectors can give long-term transduction in the rat brain without extensive inflammation, even after a subsequent peripheral immunization against the adenovirus (Schiedner et al., 1998
; Thomas et al., 2000
). Whatever strategy is chosen, it is always desirable to use a virus with a high affinity for the target tissue. A new trend in adenovirus vector construction has been to modify the tropism of the vector by swapping fibres or by introducing a new ligand in the fibre (Russell, 2000
).
The purpose of this study was to find an adenovirus serotype with tropism for human neural tumour cells. The binding and infectivity properties of Ad4p (prototype), Ad5v (vector strain), Ad11p and Ad17p (species E, C, B and D, respectively) were assessed. Ad17p was clearly an interesting candidate, since it was shown previously that recombinant Ad2 with an Ad17 fibre could infect rat neural cells more efficiently than wild-type Ad2 (Chillon et al., 1999 ). Binding properties were evaluated by flow cytometry and the ability to express viral proteins was assessed by immunostaining and 35S-labelling of proteins after infection. Binding was then analysed on the basis of CAR recognition. Computer-based models of the Ad4p and Ad17p fibre knob monomers were created from the coordinates of crystallized adenovirus fibre knobs.
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Methods |
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Virus strains and purification.
Adenoviruses used in this study were Ad4p (prototype, strain RI-67), Ad5v (vector, strain pFG140) (Graham, 1984 ; Graham et al., 1989
), Ad11p (prototype, strain Slobitski) and Ad17p (prototype, strain Ch22). Virus serotypes were all checked according to their DNA restriction patterns (Adrian et al., 1986
). Viruses were propagated in A549 cells and purified on a discontinuous CsCl gradient, as described previously (Mei et al., 1998
), to purify complete virus particles. The virion band was collected and the density was measured on a refractometer. Virions were assessed under an electron microscope. Virions were then purified from the CsCl by desalting on a NAP-10 column (Pharmacia) and eluted with 1·5 ml 10 mM PBS. Virion concentration was determined using a spectrophotometer: an OD of 1 unit (OD260-OD330) corresponds to 280 µg virions or 1012 virus particles/ml.
Virus labelling.
Virions purified from the CsCl gradient were loaded onto a NAP-10 column and eluted with 1·5 ml labelling buffer (50 mM NaHCO3 and 135 mM NaCl, pH 8·8). The virion concentration was between 1 and 4 mg/ml. MgCl2 (3 µl of a 1 M solution) was added to a final concentration of 2 mM. Then, 165 µl N-hydroxysuccinimidobiotin (1 mg/ml) (Sigma) dissolved in DMSO (Sigma) was added and the virions were mixed with biotin in the dark at 4 °C overnight. Free biotin was removed on a NAP-10 column eluted with 10 mM PBS. Glycerol was added to a 10% concentration and the virions were aliquoted and stored at -70 °C until use.
Binding experiments using a FACScan flow cytometer.
For each binding experiment, 5x105 cells were used. Cells were detached from the growth surface using PBS containing 0·05% EDTA, diluted in their respective growth medium and counted. Cells were incubated with three different concentrations of biotinylated virions: 1, 3 and 6 pg per cell (3570, 10700 and 21400 particles per cell, respectively). The experiment was then performed as described previously (Segerman et al., 2000 ). Results are presented as the mean of at least three independent experiments, with a confidence interval of 95%.
Adenovirus binding capacity to non-human cells expressing human CAR.
The ability of the viruses to bind to CAR was analysed for each of the different adenovirus serotypes. CHO-CAR cells expressing CAR and CHO-2 cells expressing
2-integrins were mixed with virions (3 and 6 pg virions per cell) of the different serotypes, as described above. Results are presented as the mean of at least three independent experiments, with a confidence interval of 95%.
Labelling of proteins after virus infection.
Pulsechase experiments were performed as described previously (Segerman et al., 2000 ). To distinguish viral proteins from cellular proteins, a mock-infected control was used for each cell line. Three independent labelling experiments were carried out and the data were consistent.
Immunostaining.
The cell lines Mg251, PFSK-1, SH-SY5Y and A549 were grown on coverslips in a 24-well microtitre plate and infected with 2 pg virions per cell (the same amount of cells were used with the different serotypes) in serum-free medium for 1 h at 37 °C. At 1 h post-infection (p.i.), medium containing 2% FBS was added and the cells were incubated for a further 24, 48 or 72 h. Cells were fixed in 2% paraformaldehyde for 30 min. A solution containing 2% BSA and 0·1% saponin in PBS (BS buffer) was added for 30 min at room temperature. Cells were then incubated for 1 h at room temperature with a mouse monoclonal antibody (mAb 8052), which recognized the hexon protein of all adenovirus serotypes used in this study (Chemicon International), diluted to 5 µg/ml in BS buffer. Cells were washed in BS buffer and then incubated for 1 h at room temperature with FITC-conjugated rabbit anti-mouse immunoglobulin G (Dakopatts), diluted 1:30 in BS buffer. The cells were washed again in PBS and mounted onto a slide. Micrographs were taken using a fluorescence microscope (Leitz Dialux 20) equipped with a Hamamatsu colour 3CCD (C5810) at a magnification of 130x.
Fibre knob alignment.
Sequence alignments were made with the MEGALIGN program (DNASTAR) using the CLUSTAL algorithm with a PAM (point-accepted mutations per 200 aligned positions) 250 residue weight table. Alignments were performed using the sequences from Protein Database (PDBnr): accession numbers s39300 for the Ad4p fibre, ERAD5 for the Ad5 fibre, D37476 for the Ad11p fibre and UNK_52178293 for the Ad17 fibre.
Ad4p and Ad17p fibre knob modelling.
The Ad4p knob monomer model was created with the SWISS-MODEL ProMod II system (Guex & Peitsch, 1997 ; Guex et al., 1999
; Peitsch, 1995
) using the X-ray crystallographic coordinates of the Ad5 fibre knob (Xia et al., 1994
) and the corresponding alignment of Ad4p (accession number s39300). The Ad17p fibre knob monomer was modelled based upon the crystallographic coordinates for the Ad2, Ad5 and Ad12 fibre knobs (accession numbers 1QHVA, 1KNB and 1NOBD, respectively) and the corresponding alignment of Ad17p (accession number UNK_52178293). Structures were refined using Swiss-PdbViewer with respect to energy minimization and the molecular surface was computed. The rootmeansquare (RMS) value of the Ad4p fibre knob monomer model versus the crystallized Ad5 was 0·55
. The RMS value for the Ad17p fibre knob monomer was 1·17
. Surface residues with no similarity to Ad5 were coloured blue and the positions of conserved CAR-interacting residues were coloured green. White regions represent amino acid residues also present in Ad5 (see Fig. 4) (Bewley et al., 1999
; Roelvink et al., 1999
).
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Results |
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Ad4p and Ad11p were expressed early in the neural cell lines
Binding does not necessarily lead to internalization and expression of viral proteins. An immunostaining protocol was performed to assess whether the different serotypes could also become internalized and start transcription and translation of their genes in these cell lines (Fig. 5). Cultures were analysed for hexon expression at three time-points p.i., showing that the time of virion expression in the cells was dependent both on the cell line and on the serotype of the virus. The glioblastoma cell line Mg251 was permissive for Ad4p, Ad5v and Ad11p infection but refractory to Ad17p, where, only occasionally, a cell could be scored positive 72 h p.i. Viral hexon proteins of Ad5v, Ad11p and Ad17p could not be detected in the glioblastoma cell line 24 h p.i., when Ad4p hexon was produced in a low percentage of the cells (Fig. 5a
). After 48 h, the proportion of Ad4p-, Ad5v- and Ad11p-infected cells had increased to a substantial majority of the cells. Ad17p did not show any hexon production 48 h p.i. in this cell line. In the medulloblastoma cell line PFSK-1, a few cells scored positive 24 h after infection with both Ad4p and Ad11p, indicating an efficient internalization and expression in these cells. At 72 h p.i., both Ad4p and Ad11p manifested a high proportion of positive cells. Ad5v was expressed but to a lesser extent than Ad4p and Ad11p in PFSK-1 cells. Ad17p showed a refractory pattern with only a few positive cells at 72 h p.i. (Fig. 5b
). The neuroblastoma cell line SH-SY5Y was only semipermissive to infection by the different serotypes. Ad4p was the only serotype expressed 24 h p.i. but the number of positive cells was fewer than after infection of the other two cell lines. At 48 h p.i., only Ad4p and Ad11p manifested infection in SH-SY5Y cells, Ad17p did not infect the cells at all, while Ad5v showed some expression 72 h p.i. Ad4p infected the highest proportion of cells in the neuroblastoma cell line; moreover, Ad4p and Ad11p displayed the most rapid expression (Fig. 5c
).
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Discussion |
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The data presented in this report support the notion that Ad4p of species E has affinity for CAR, as has been described previously (Roelvink et al., 1998 ). However, Ad4p was, as shown here, more efficient in infecting neural tumour cells than Ad5v. Furthermore, Ad4p bound with a higher efficiency to CAR on CHO-CAR cells than Ad5v. A difference in the binding characteristics between these two CAR-binding viruses was not surprising. There are some major differences in the fibre knob between Ad4p and Ad5v. They are both binding to the same receptor, but the amino acid sequence reveals a 33% dissimilarity in the fibre knob between these serotypes (Chroboczek et al., 1995
), which may have affected the docking on CAR. A structure model of the fibre knob of these viruses showed that a large part of this dissimilarity was located in an area bordering the conserved CAR-binding region. Possibly, this region was responsible for the difference in binding properties seen between Ad4p and Ad5v. Other aspects, such as differences in surface-exposed charges, could increase electrostatic repulsion by the negatively charged glycocalyx. Also, the length of the fibre has been shown to influence binding. A short fibre will facilitate direct binding between the penton base and
v-integrins on the cell but could also make the virion sensitive to electrostatic interactions between the hexon and cell surface (Shayakhmetov & Lieber, 2000
). Ad4p belongs to species E but has characteristics from both species B and C. The early gene sequences of Ad4p show a large resemblance with species B adenoviruses, while the fibre knob gene is similar to species C adenoviruses. The length of the fibre is an intermediate between species B and C (Gruber et al., 1993
).
The fibre knob of Ad4p, species E, showed a higher similarity to Ad5 of species C than the fibre knob of Ad17p of species D did. There was only 48, 46 and 37% identity between the Ad17p fibre knob and the CAR-binding fibre knobs of Ad5, Ad2 and Ad12, respectively, which were used in the modelling of the Ad17p knob. In the model structure of Ad17, there were dissimilarities adjacent to the CAR-binding residues and also a large area of divergence in the regions of the CD-, GH- and IJ-loops (Fig. 4b, blue region on the left) (van Raaij et al., 1999
). By modelling, we showed that this region may be easily accessible on the fibre knob and the observed structural difference could account for the differences in the binding characteristics between these serotypes. This question can be further investigated by point mutation analysis of this region in the fibre knob. The presence of the conserved CAR-binding residues in the AB-loop in Ad17p does not necessarily provide a good affinity for CAR. It has been shown that other species D adenoviruses, like Ad37, contain these residues but use sialic acid as a primary receptor instead of CAR (Arnberg et al., 2000a
, b
). It has also been suggested that Ad17p has receptors other than CAR or MHC-1 for attachment to cells (Zabner et al., 1999
). The fibre knob of Ad11p shared only 31 % identity with the species C Ad5 fibre knob and could not be properly modelled based on the crystal structure of Ad5.
Ad11p is a very interesting vector candidate because of its high binding capacity towards neural cell lines. The ability to infect a cell population is initially dependent on affinity for the particular cell type. Actions that enhance virus binding will increase adenovirus-mediated gene transfer (Fasbender et al., 1997 , 1998
; Kaplan et al., 1998
; Toyoda et al., 1998
). However, the affinity required for efficient transfection of a cell population may be different for different serotypes and cell lines. Also, the stages between internalization and expression of the viral proteins are important. Different serotypes have different strategies before entering the nucleus, which may influence the success rate of infecting different cell types. Species B and C viruses have an equally rapid internalization (t1/2=23 min) but intracellular trafficking is different and seems to be dependent on the adenovirus fibre (Miyazawa et al., 1999
). Adenoviruses of species C are quickly released to the cytoplasm from the early endosome and translocated to the nucleus along microtubules. Members of species B have chosen another route and remain encapsidated until they reach the late endosome or lysosome (pH 5·5 and 5·0, respectively) before transport to the nucleus (Miyazawa et al., 2001
).
The biodistribution of CAR in the body seems to be dependent on the stage of cell differentiation. In the CNS, CAR is suggested to be implicated as a cell adhesion molecule in neural network formation of the developing brain (Honda et al., 2000 ). In the adult brain, expression is more restricted and limited to the ependymal cells lining the ventricular system (Tomko et al., 2000
). Other areas of the body also show a developmental restriction of CAR expression. CAR is not expressed on primitive CD34+ and CD38- haematopoietic progenitor cells but expression is acquired during erythroid and myeloid differentiation (Rebel et al., 2000
).
CAR expression in different glioma cell lines is highly variable (Asaoka et al., 2000 ; Miller et al., 1998
) and the transfection efficiency with Ad5 vectors is correlated to the CAR content on the cell (Asaoka et al., 2000
; Mori et al., 1999
). Because of this, alternative non-Ad5 serotypes should be developed for gene therapy of neural tumours. The non CAR-binding serotype Ad11p showed a much higher affinity for all of the tested neural cell lines than Ad5v and is therefore a very interesting candidate vector for tumour cells of neural origin.
High expression of Ad4p, Ad5v and Ad11p viral proteins in the glioblastoma cell line was noted in the 35S-labelling experiment. In the medulloblastoma cell line there was only efficient expression of Ad4p and Ad11p, which was supported by immunostaining experiments in which Ad4p and Ad11p showed the largest and quickest response. The lower Ad5v expression, as visualized by immunofluorescence, was not sufficient to generate visible bands during SDSPAGE. Only Ad4p produced sufficient amounts of viral proteins in the neuroblastoma cell line. It was interesting to see that Ad4p, despite a low binding capacity for the neural cell lines, could infect a relatively large proportion of cells. Under these experimental conditions, a relatively low binding capacity was demonstrated for Ad4p. However, an efficient expression was detected using both SDSPAGE and immunofluorescence, making Ad4p an important vector candidate. Virus expression of all the serotypes tested in the neuroblastoma cell line (SH-SY5Y) was very slow. The immunostaining procedure showed that the virus types could infect the neural cell lines but only to a varying degree and with a different time span from infection to virus expression. The efficiency of Ad17p was very poor in all of the neural cell lines tested. The two vector candidates Ad4p and Ad11p seem to be more efficient than the currently used Ad5. A slow response in the production of structural viral proteins normally results from a low m.o.i. for the virus serotype on that particular cell line (Fenner et al., 1974 ). It has also been shown that the length of virus incubation is often more important than the m.o.i. for the infection success rate. A longer incubation time with virions results in a higher transduction efficiency (Marini et al., 2000
). Ad4p showed the most rapid response in all of the cell lines and Ad11p also manifested a rapid and potent expression capacity in all of the cell lines.
The large number of adenovirus serotypes and the fact that different adenoviruses have distinctive tropism (Wadell, 1999 ) give an indication that different adenovirus candidate vectors could be evaluated for different tissues. Another important issue when choosing a suitable vector is the prevalence of immunity against the adenovirus vector candidate and, in this respect, the choice of the endemic Ad2 and Ad5 is not optimal. The percentage of adults with neutralizing antibodies against Ad2 and Ad5 is very high (88 and 68%, respectively) and the fact that the viruses are endemic gives ample possibilities for boosting the titre (Fox & Hall, 1980
; Kremer et al., 2000
; Piedra et al., 1998
; Wadell, 1999
). About 34% of adults have neutralizing antibodies against the epidemic Ad4 (Ludwig et al., 1998
). The seroprevalence in adults for Ad11 and Ad17 is not found in the literature. The high prevalence of immunity against Ad2 and Ad5 will impair long-term expression of most vectors based on these serotypes. This also applies to virus expression in the CNS when the recipient can be challenged with the common Ad2 or Ad5 infection (Thomas et al., 2000
).
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Acknowledgments |
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Received 2 October 2001;
accepted 24 January 2002.