E1-deleted adenovirus (Ad) vectors expressing the human to the induction of a cellular immune response to the lacZ coagulation factor IX (hFIX) or the bacterial ␤-galactoantigen. In contrast, absence of detectable levels of serum sidase (lacZ) were injected intravenously into various hFIX in immunocompetent animals was not associated with strains of immunocompetent (C57Bl/6, BALB/c, CD1, a loss of viral DNA but was strictly correlated with the CBA/J, C3H) and immunodeficient (BALB/c-nu/nu, induction of anti-hFIX antibodies. Surprisingly, anti-hFIX C57Bl/6-nu/nu, SCID, NIH-bg-nu-xid) mice. Regular analyantibodies were never detected in C57Bl/6 mice, leading sis of mouse sera and tissues showed a persistent to prolonged detection of hFIX. These results suggest that expression of both transgenes in immunodeficient mice, cellular immunity to viral antigens plays a minor role in the while detection diminished very rapidly in immunocompetearly extinction of transgene expression and illustrate the ent mice. The mechanisms responsible for the transient influence of the cellular (eg lacZ) or humoral (eg hFIX) detection of the two transgenes were however not identimmunity to transgene-encoded products on the persistical. Rapid decline of lacZ expression was correlated with a ence of transgene expression. rapid decrease of viral DNA sequences, and consequently

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The coxsackievirus and adenovirus receptor (CAR) is a high affinity receptor used by adenoviruses, including adenovirus type 5 (Ad5). The adenovirus fibre molecule bears the high affinity cell binding domain of Ad5, allowing virions to attach to CAR. The avian adenovirus CELO displays two fibre molecules on its capsid and it was logical to expect that the cell binding functions of CELO might also reside in one or both of these fibres. We had previously shown that the cell binding properties of CELO resemble Ad5, suggesting that the two viruses use similar receptors. Experiments with CAR-deficient CHO cells and CHO cells modified to express CAR demonstrated that CELO has CAR-dependent transduction behaviour like Ad5. Mutations were introduced into the CELO genome to disrupt either the long fibre 1 or the short fibre 2. A CELO genome with fibre 2 disrupted did not generate virus, demonstrating that fibre 2 is essential for some stage in virus growth, assembly or spread. However, a CELO genome with disrupted fibre 1 gene produced virus (CELOdF1) that was capable of entering chicken cells, but had lost both the ability to efficiently transduce human cells and the CAR-specific transduction displayed by wild-type CELO. The ability of CELOdF1 to transduce chicken cells suggests that CELOdF1 may still bind, probably via fibre 2, to a receptor expressed on avian but not mammalian cells. CELOdF1 replication was dramatically impaired in chicken embryos, demonstrating that fibre 1 is important for the in vivo biology of CELO.

A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.To successfully infect cells, adenoviruses must reach the nucleus, where viral gene expression can begin and the genomes can replicate. Adenoviruses are nonenveloped; the viral particle consists of an icosahedral protein capsid surrounding the 36-kb linear double-stranded DNA genome and associated DNA binding proteins (reviewed in reference 40). Human adenoviruses infect predominantly epithelial cells and can trigger respiratory and gastrointestinal tract ailments of a mild course in the majority of cases (reviewed in reference 23

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.