Abstract
Most adenovirus (Ad) vectors are E1 gene deleted replication defective (RD-Ad) vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad) vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad) vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP) expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG) cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In immunodeficient mice, SC-Ad drove stronger luciferase expression than RC- or RD-Ad. These data demonstrate better transgene expression by SC- and RC-Ad in vitro and in vivo than RD-Ad. This higher expression by the replicating vectors results in a peak of expression within 1 to 2 days followed by cell death of infected cells and release of transgene products. While SC- and RC-Ad expression were similar in mice and in Syrian hamsters, RC-Ad provoked much stronger ISG induction which may explain in part SC-Ad′s ability to generate stronger and more persistent immune responses than RC-Ad in Ad permissive hamsters.
Highlights
Recombinant human adenoviral (Ad) vectors have shown promise as oncolytic and vaccine
To better understand how the vectors function as gene expression platforms, we examined here the interplay between transgene expression, the cellular reaction to infection, and cell death mediated by RD, SC, and replication-competent Ad (RC-Ad) vectors in vitro and in vivo
In our previous work characterizing single cycle adenoviruses, we have shown that increases in green fluorescent protein (GFP) and luciferase expression directly correlate with the ability of the virus to replicate its genome both in vitro and in vivo
Summary
Recombinant human adenoviral (Ad) vectors have shown promise as oncolytic and vaccine. There is renewed interest in the use of Ad vectors expression could their enhance transgene-directed immune responses and increase efficacy in vaccines that can replicate genome, because their ability to amplify transgene expression could enhance and in cancer therapies [3,4,5,6]. Only SC-Ad generated stronger immune responses against transgene antigen While this was expected for RD-Ad, we were surprised that SC-Ad was better than RC-Ad considering that RC-Ad had the possibility for a second cycle (or more) of infection and transgene expression. This observation is not unique to single-cycle Ad, but has been observed when comparing single-cycle and replication-competent flavivirus vectors [8]. To better understand how the vectors function as gene expression platforms, we examined here the interplay between transgene expression, the cellular reaction to infection, and cell death mediated by RD-, SC-, and RC-Ad vectors in vitro and in vivo
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