P037 Effects of vaccinia virus-expressed IRF-3 ON VACV replication in tissue culture and in atopic Nc/Nga mice

Abstract

Introduction Vaccinia viruses (VACV) of several strains had been used in the eradication campaign against smallpox. Recently, vaccination of selected groups of professionals was reintroduced in certain countries, imposing a risk of an inadvertent spread of VACV to non-immune population. In individuals with a history of atopic dermatitis (AD), inoculation of VACV can result in eczema vaccinatum, an uncontrolled spread of VACV in the skin. Pathogenesis of eczema vaccinatum has not been completely understood, but Th2-skewed immune responses together with lower immigration of immune cell types, such as NK cells, plasmacytoic dendritic cells and neutrophils, and defective cytokine production and antimicrobial peptides have been suggested. Recently, increased production of IL-17 resulting in lower immigration of NK cells and increased VACV replication were demonstrated. VACV encodes many factors that interfere with host immune responses. Among others, VACV specifically inhibits phosphorylation of IRF-3 and expression of type I interferons at multiple levels. To overcome this block, we have generated a recombinant VACV expressing murine IRF-3. Methods We have cloned murine IRF-3 cDNA into pSC11, a vector expressing the recombinant gene under control of VACV early/late promoter p7.5 and allowing homologous recombination into the thymidine kinase gene of VACV. The recombinant VACV expressing IRF-3 (VACV-IRF3) was selected, and the insertion and expression of IRF-3 were confirmed by Southern blot analysis and RT-PCR, respectively. The growth of the virus in green monkey kidney derived BSC-40 cells and in mouse fibroblasts NIH-3T3 is characterized by serial dilutions and plaque assays, changes in host cell morphology by optical and fluorescent microscopy, expression of murine IFN-beta by RT-PCR. The growth of the virus and immune responses towards it are characterized in atopic Nc/Nga mice, using serial dilutions and plaque assays, ELISA and neutralization assays. Results Murine IRF-3 mRNA was found expressed by VACV-IRF3 in cells of different embryonic origins. Based on preliminary experiments, its expression does not seem to affect virus yields in tissue culture in comparison with VACV expressing luciferase (VACV-LUC). However, VACV-IRF3 seems to induce expression of murine IFN-beta and apoptosis in mouse NIH-3T3 fibroblasts while causing lytic infection of monkey BSC-40 cells. A study of the effects of IRF-3 expression on virulence, size of the lesions after a skin inoculation, and the titers of VACV-specific antibodies in the atopic Nc/Nga mice is currently under progress. Conclusion Expression of murine IRF-3 by a VACV recombinant seems to overcome VACV-mediated inhibition of IFN type I expression in murine cells. Increased production of IFN type I could decrease a risk of eczema vaccinatum and other post-vaccination complications that result from uncontrolled VACV growth. Apoptosis of VACV-IRF3-infected cells could affect mounting of VACV-specific immune responses.