The innate immune response to intraportally infused adenoviral vector was evaluated in rhesus monkeys. A first-generation adenovirus-expressing lacZ (Ad-lacZ) was administered at a dose just below that which causes severe morbidity. The response to vector was evaluated for the initial 24 h following infusion. Clinical findings during this time were primarily limited to petechiae, consistent with the development of thrombocytopenia and biochemical evidence of disseminated intravascular coagulation. Serum transaminases were elevated and a lymphopenia developed. Tracking of fluorescent-labeled vector demonstrated distribution to macrophages and dendritic cells of the spleen and Kupffer cells of the liver. A systemic release of the cytokine IL-6 occurred soon after vector infusion. Analysis of splenic cells revealed acute activation of macrophages and dendritic cells followed by massive apoptosis. Bone marrow cultures demonstrated normal erythroid and primitive progenitors with a significant decrease in myeloid progenitors. Similar findings, except the abnormality in bone marrow cultures, were observed in monkeys who received an identical dose of Ad-lacZ in which vector genes were inactivated with psoralen and UV irradiation. These data suggest that inadvertent targeting of antigen-presenting cells following intraportal infusion of vector leads to a systemic cytokine syndrome which may be triggered by the viral capsid proteins.

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. Flavivirus virions contain single-stranded positive-sense RNA of ϳ11 kb encapsidated by the structural proteins C, prM, and E (18, 19). The mechanism ensuring selective packaging of only the flavivirus RNA into virions in virus-infected cells, as well as the required signals in the RNA and in the structural proteins involved in this process, have not been determined. We demonstrated previously in trans-encapsidation experiments using Kunjin virus (KUN) replicon RNA with deleted structural genes that only KUN replicon RNA was packaged into the secreted virus-like particles, while coreplicating Semliki Forest virus replicon RNA (used as a vector for expression of KUN structural genes) was not packaged (6). In addition, our trans-complementation experiments with KUN genomic RNAs containing deletions in the NS1 and NS5 genes, and trans-complementation experiments of others with yellow fever virus RNAs containing deletions in the NS1 gene, both using as helpers Sindbis virus replicons expressing corresponding wild-type flavivirus nonstructural genes, showed that only the flavivirus RNAs and not the coreplicating Sindbis virus replicon RNAs were packaged into secreted virions by the flavivirus structural proteins (9,11,12). These trans-encapsidation and trans-complementation experiments demonstrated clearly that packaging of flavivirus RNA occurs by a highly specific mechanism.Our encapsidation studies with KUN replicon RNAs also showed that the most efficient packaging of replicon RNA into virus-like particles occurred at the time of maximum RNA replication (6, 16), suggesting that these two processes (replication and packaging) are closely related. Similarly, in flavivirus-infected cells the assembly and release of infectious virions coincided with the large increase in viral RNA synthesis at the end of the latent period (18). Interestingly, coupling between replication and packaging of poliovirus RNA as a mechanism ensuring its specific encapsidation was initially proposed by Baltimore (1) and recently demonstrated by Nugent et al. (15). The latter study showed that selective inhibition of replication of poliovirus replicon RNA by guanidine dramatically decreased the encapsidation efficiency of the accumulated replicon RNA by the poliovirus capsid proteins provided in trans by the coinfected guanidine-resistant mutant poliovirus. It was suggested by the authors that only actively replicating RNA (i.e., an RNA strand emerging from the replication complex) could be encapsidated by the structural proteins.Since no selective inhibitors of flavivirus RNA replication have been reported, we decided to take a different approach to stud...

Development of gene expression vectors based on subgenomic replicons of positive-strand RNA viruses has gained much attention over the last decade (11,32). Genomes of the alphaviruses Semliki Forest virus (SFV) (7, 29), Sindbis (SIN) virus (1, 10, 15) and Venezuelan equine encephalitis virus (12, 37), as well as the poliovirus genome (34, 36), have all been used. An important characteristic feature of these systems is the ability of replicon RNA to self-replicate, thereby amplifying the input template in the host cell. This amplification in turn leads to increased production of encoded proteins. Replicon RNAs can be delivered into host cells by direct transfection with RNA transcripts produced in vitro from corresponding plasmid DNAs (20,47) or by infection with virus-like particles (VLPs) containing encapsidated replicon RNAs (10,12,29,37). Alternatively, they can be transcribed from transfected replicon-encoded plasmid DNAs utilizing cellular RNA polymerase II transcription machinery (1,7,14,15). It was shown that replicon-based DNA vectors produced higher levels of encoded heterologous proteins than conventional plasmid DNA expression vectors and also elicited greatly enhanced immune responses (7,19).Applications for most of the alphavirus and poliovirus replicon vectors have been limited to only short-term transient expression due to the cytopathic effects (CPE) induced by vector replication in mammalian cells (18). To address this problem, noncytopathic SIN virus replicon-based vectors containing the puromycin resistance gene were developed by isolation of SIN replicon mutants adapted to puromycin selection in BHK cells (1,17). However, the use of these vectors is restricted by a number of limitations, such as a narrow host range, relatively low levels of heterologous gene (HG) expression, and some instability of expression in cell populations during passaging (1).We have been developing a gene expression system based on subgenomic replicons of another RNA virus, the flavivirus Kunjin (KUN), containing deletions in the structural region of the genome (25). In contrast to the alphavirus and poliovirus replicons, as well as full-length KUN RNA, replication of KUN replicons in mammalian cell cultures did not produce any apparent CPE (25). Recently we reported the construction and use of RNA-based KUN replicon vectors for HG expression in cell culture after the direct transfection of in vitrosynthesized recombinant KUN replicon RNAs or after infection with recombinant KUN VLPs (43). In this study we describe the development of DNA-based KUN replicon vectors and demonstrate their ability to direct high-level prolonged HG expression in a range of cell lines and in vivo. Moreover, we show the induction of antibody response against a KUN vector-encoded HG after immunization of mice with the corresponding KUN replicon DNA construct. These noncytopathic DNA-based KUN replicon expression vectors should be useful for a variety of applications both in vitro and in vivo.