This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Variola virus and vaccinia virus contain a large number of immunoregulatory gene products which are thought to enhance viral reproduction by modulating host immune responses. Secretion of interferon-gamma (IFNgamma) is a commonly used measure of cellular immune responses in vitro. We hypothesized that the vaccinia virus B8R protein, a secreted IFNgamma-receptor analog, might interfere with detection of IFNgamma by the ELISPOT assay. IFNgamma secretion by peripheral blood mononuclear cells (PBMCs) in response to stimulation with vaccinia virus or autologous B cells transformed by Epstein-Barr virus (EBV) was quantitated by ELISPOT. Vaccinia infection inhibited CD8+ T cell IFN-gamma ELISPOT responses to autologous EBV-transformed B cell lines by 70%. In contrast, the EBV-specific IFN-gamma ELISPOT response was not inhibited by infection with modified vaccinia Ankara (MVA), an attenuated vaccinia strain that does not contain B8R. Stimulation of PBMCs with a vaccinia virus recombinant that has had the B8R gene deleted resulted in a 75% increase in detection of IFNgamma secretion compared with the wild-type strain. Stimulation of PBMCs from vaccinia vaccinees with MVA resulted in a 12-fold increase in IFNgamma secretion compared with the wild-type strain. Vaccinia virus immunoregulatory gene products interfere with in vitro detection of cellular immune responses, including vaccinia-specific and EBV-specific responses. Deletion of the soluble IFN-gamma receptor (B8R) from vaccinia virus resulted in an incomplete abrogation of this effect, suggesting that other genes absent in MVA also contribute to evasion of CD8+ T cell mediated immune responses. These results suggest that use of vaccinia virus as a stimulus to assess IFNgamma secretion may underestimate the host immune response
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