Poxviruses such as the causative agent of smallpox have developed multiple strategies to suppress immune responses, including the suppression of DC activation. Since poxviruses are large DNA viruses, we hypothesized that their detection by DCs may involve the endosomal DNA recognition receptor TLR9. Indeed, we have shown here that DC recognition of ectromelia virus (ECTV), the causative agent of mousepox, completely depended on TLR9. The importance of TLR9 was highlighted by the fact that mice lacking TLR9 showed drastically increased susceptibility to infection with ECTV. In contrast, we found that the strongly attenuated poxvirus modified vaccinia virus Ankara (MVA) activated DCs by both TLR9-dependent and -independent pathways. We therefore tested whether we could use the broader induction of immune responses by MVA to protect mice from a lethal infection with ECTV. Indeed, MVA given at the same time as a lethal dose of ECTV protected mice from death. Importantly, MVA also rescued TLR9-deficient mice if administered 2 full days after an otherwise lethal infection with ECTV. Therefore, these data suggest an essential role for TLR9 in the defense against poxviruses. In addition, postexposure application of MVA may protect against lethal poxvirus infection.
These results suggest that the same mechanisms controlling gene transcription of the host cell's genome are active in controlling transgene expression and that this should be taken into account when constructing vectors for gene therapy. The assay reported in this study could be used as a screening method to evaluate new vectors.
Here we describe a PCR-based analysis system that allows the simple simultaneous assessment of murine interferons (IFN)-alpha and IFN-beta induction in a single reaction. In this analysis, the so-called early IFN-alpha4 can be distinguished from the so-called late IFN-nonalpha4 by employing a primer mixture that amplifies a part of the IFN-alpha genes in which IFN-alpha4 exhibits a deletion of 15 nucleotides compared to IFN-nonalpha4. By including a final denaturation and a slow cooling step at the end of the PCR procedure, hybrid formation was avoided that regularly occurred when standard protocols were used. Separation of the amplification products on 4.5% agarose gels allowed the comparative assessment of the classical type I IFNs. Using this analysis system, we could show that in immortalized adult fibroblasts, IFN-beta is induced first and the two types of IFN-alpha are induced later and simultaneously. When similar fibroblasts derived from mice that lack IFN-beta were tested, the IFN response was delayed. However, now IFN-alpha4 appeared first and apparently induced the cascade of IFN-nonalpha4. This confirms the role of IFN-beta as master regulator of the normal IFN response.
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