Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.