Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vectorencoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10 7 infectious units͞ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replicationcompetent virus, suggesting utility for production of largescale vector preparations. Furthermore, both Sindbis virusbased and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirusderived vectors.The use of virus-derived expression vectors for gene therapy and vaccine applications increasingly is being pursued, with a number of diverse virus types and approaches. Alphaviruses are attractive candidates for such applications because of their high levels of replication and gene expression, their ability to infect a variety of diverse cell types, and the ability to manipulate cDNA clones from which infectious viral RNA may be transcribed (for review, see refs. 1 and 2). The alphavirus genome is a single-stranded, positive-sense RNA of approximately 11.7 kb and is encapsidated within an icosahedral capsid protein shell (for review, see ref.3). Nucleocapsids, in turn, are surrounded by a host-derived lipid envelope from which the viral spike glycoproteins E1 and E2 protrude. Cytoplasmic replication of the RNA genome is mediated by four viral-encoded nonstructural proteins and proceeds through a full-length negative-sense intermediate. Subsequent positive-strand RNA synthesis results in both progeny genome RNA and an abundant, internally initiated subgenomic mRNA. The virus structural proteins are translated from the subgenomic mRNA as a polyprotein that is processed into the individual components of the virion.The general strategy for construction of alphavirus-based expression vectors has been to substitute the viral structural protein genes with a heterologous gene, maintaining transcriptional control via the highly active subgenomic RNA promoter (1, 2, 4). As such, these vector r...

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.

The mechanism of immunosuppression induced by bovine herpesvirus 1 (BHV-1) was investigated by studying the effects of the virus on the expression of major histocompatibility complex (MHC) class I molecules. After infection with the virus, the expression of class I molecules was detected by flow cytometry and pulse-chase analysis. A selective downregulation of expression of class I molecules was seen in the infected cells, while the class II expression remained unaffected. The reduction in surface expression was evident as early as 8 hours postinfection, reaching significant levels by 12 hours. The downregulation was seen with a multiplicity of infection as low as 0.1. A modified live vaccine strain of BHV-1 also induced the downregulation of class I expression. Analysis of the viral proteins(s) involved in this downregulation with metabolic inhibitors (cycloheximide or phosphonoacetic acid), suggested that the immediate early and/or early proteins of the virus mediate this effect. Pulse-chase analysis revealed that the synthesis of the class I heavy chain, and the assembly/transport of class I molecules were affected by the virus infection. These results suggest that BHV-1 interferes with the molecular mechanisms involved in the synthesis, and assembly/transport of MHC-class I molecules. This interference with the class I antigen processing pathway might help the virus to evade the cytotoxic T-lymphocyte response of the host.

The objective of this study was to investigate the effect(s) of bovine herpesvirus 1 (BHV-1) infection on the expression of MHC class I molecules in murine fibroblasts. L-cells were infected with BHV-1 at a multiplicity of infection (m.o.i.) of 10 plaque forming units (PFU) per cell, and the expression of MHC class I molecules was analyzed by flow cytometry and immunoprecipitation. Temporal studies revealed a reduction in class I expression beginning at 8 h post infection (p.i.) which reached a maximum between 10 to 16 h p.i. The loss of class I expression was restored in the presence of phosphonoacetic acid (30 micrograms/ml), a late herpesviral protein synthesis inhibitor. However, addition of cycloheximide, a total protein synthesis inhibitor (100 micrograms/ml), did not result in any difference in class I expression between virus-infected and mock-infected cells. These results suggest that the reduced class I expression is a direct consequence of BHV-1 infection, and that the late viral gene product(s) may be involved in this process. Similar phenomena may occur in natural BHV-1 infection in cattle, and this may be one of the mechanisms of immune suppression by BHV-1.

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