The Childrens Hospital of Philadelphia, Division of Immunologic and Infectious Diseases, Abramson 1202, 3516 Civic Center Boulevard, Philadelphia, PA 19104, USA1
The Wistar Institute, Philadelphia, PA 19104, USA2
The Institute for Human Gene Therapy, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA3
Author for correspondence: Christopher Cohen. Fax +1 215 590 2025. e-mail Cohenc{at}email.chop.edu
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Abstract |
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Introduction |
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Efficient gene delivery is inhibited by preexisting immunity to virus vectors (Xiang et al., 1999 ) and most adults have measurable titres of antibodies capable of neutralizing Ad2 and -5 (Farina et al., 2001
). To circumvent this problem, several nonhuman adenoviruses have been adapted as vectors for gene delivery (Soudais et al., 2000
; Tan et al., 2001
). A replication-defective vector based on chimpanzee adenovirus type 68 (C68) has been developed recently (Farina et al., 2001
). Experiments in animals have demonstrated that peptides delivered in the C68 vector can elicit both humoral and cellular immune responses (Xiang, Z. Q., Gao, G., Reyes-Sandoval, A., Cohen, C. J., Li, Y., Bergelson, J. M., Wilson, J. M., Ertl, H. C. J., unpublished results). Because neutralizing antibodies to common adenovirus serotypes do not cross-neutralize C68 (Basnight et al., 1971
) and because human adults have no preexisting immunity to C68 (Farina et al., 2001
), this chimpanzee-derived vector may have significant advantages both as a vaccine vector and for therapeutic gene delivery.
Clinical applications of C68 vectors will depend on understanding the interactions between the virus and its target cells and tissues. Identification of the receptor for C68 can provide important information about how this vector may behave in vivo. We have used several approaches to demonstrate that CAR is the primary receptor for C68 on a number of cell lines. Thus, it is likely that knowledge obtained about Ad2 and -5 tropism will, at first approximation, be relevant to understanding the behaviour of C68.
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Methods |
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For infection with adenovirus, 5x105 cells were seeded into 6-well tissue culture dishes and cultured overnight. The next day, tissue culture medium was removed and replaced with 1 ml Hanks balanced salt solution containing 10 mM MgCl2, 10 mM HEPES buffer, 4 mM CaCl2 and 4% FCS. Cells were incubated with C68, Ad5 or -7, all encoding green fluorescent protein (GFP), at an m.o.i. of 1 p.f.u. per cell for 1 h at room temperature with gentle rocking. Virus was removed, cells were washed once with PBS, medium was replaced and cells were incubated at 37 °C. After 48 h, GFP expression was determined either by flow cytometry or by examination with a Nikon Eclipse 800 fluorescent microscope. All experiments were performed at least three times.
The hCAR extracellular domain was produced as a soluble immunoglobulin Fc fusion protein, as described previously (Martino et al., 2000 ). To test if soluble CAR blocked infection of CHO-hCAR cells by Ad5 or C68, virus was incubated with or without 5 µg of soluble CAR for 1 h at room temperature before the addition of virus to the cells. Polyclonal anti-hCAR rabbit serum was raised against the His-tagged CAR extracellular domain produced in a baculovirus system. CHO-hCAR, CHO-mCAR or HeLa cells were preincubated with a 1:100 dilution of either the rabbit polyclonal anti-hCAR antiserum or preimmune serum for 1 h at 37 °C prior to the addition of virus.
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Results |
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Discussion |
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A variety of other molecules may function in adenovirus attachment or entry into cells. Integrins, including V
3 and
V
5, facilitate virus internalization by a mechanism that involves recognition of an RGD motif within the viral penton base protein (Wickham et al., 1993
); the C68 penton base, like those of Ad2 and -5, includes an RGD motif, suggesting that integrins may play a role in internalization by C68. For some adenoviruses, or on some cell types, attachment to other receptor molecules may also be important for infection (Arnberg et al., 2000
; Hong et al., 1997
; Huang et al., 1996
) and alternative receptors may influence the tropism of C68. However, the inhibition of transduction by a CAR-specific antibody suggests that, at least for HeLa and CHO cells, any CAR-independent entry pathways must, at best, be inefficient.
C68 is serologically distinct from all human adenoviruses tested (Basnight et al., 1971 ; Farina et al., 2001
), but its fibre protein is closely related to that of human Ad4 and identical within the region of the knob domain implicated in fibre interaction with CAR. Thus, it is likely that the mechanism of receptor attachment by C68 is similar to that for other CAR-binding viruses. The fibres of Ad4 and C68 (426 aa) are shorter than those of Ad2, -5 and -12 (580590 aa). It has been suggested that the very short Ad9 fibre (362 aa) may permit the direct interaction between secondary receptors, such as integrins, to recognition sites on the virus (Roelvink et al., 1996
; Shayakhmetov & Lieber, 2000
). However, Ad4 attachment itself depends exclusively on fibre interaction with CAR (Roelvink et al., 1998
) and this is likely to be true for C68 as well.
Although a variety of factors may influence C68 interaction with cells and tissues, identification of CAR as a receptor for this virus provides an intellectual framework for empirical studies of virus tropism. The considerable literature on CAR-dependent gene delivery by Ad2 and -5 can inform experimental approaches and a variety of CAR-specific reagents can be applied to understanding the tropism of this new vector.
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Acknowledgments |
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References |
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Received 10 July 2001;
accepted 19 September 2001.