ANALYSIS OF IMMUNOREGULATORY VACCINIA GENES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 天花病毒和牛痘病毒含有大量的免疫调节基因产物，其被认为通过调节宿主免疫应答来增强病毒繁殖。干扰素-γ（IFN γ）的分泌是体外细胞免疫应答的常用量度。我们假设牛痘病毒B8 R蛋白（一种分泌型IFN-γ受体类似物）可能干扰ELISPOT检测IFN-γ。通过ELISPOT定量外周血单核细胞（PBMC）响应于用牛痘病毒或由EB病毒（EBV）转化的自体B细胞刺激的IFN γ分泌。牛痘感染抑制CD 8 + T细胞IFN-γ ELISPOT对自体EBV转化的B细胞系的应答达70%。相反，EBV特异性IFN-g ELISPOT应答不受修饰的安卡拉牛痘（MVA）（一种不含B8 R的减毒牛痘株）感染的抑制。用B8 R基因缺失的重组痘苗病毒刺激PBMC，与野生型毒株相比，IFN γ分泌的检测增加了75%。用MVA刺激来自牛痘接种者的PBMC导致IFN γ分泌比野生型菌株增加12倍。牛痘病毒免疫调节基因产物干扰细胞免疫应答的体外检测，包括牛痘特异性和EBV特异性应答。从牛痘病毒中删除可溶性IFN-γ受体（B8 R）导致这种效应的不完全消除，表明MVA中不存在的其他基因也有助于逃避CD 8 + T细胞介导的免疫应答。这些结果表明，使用牛痘病毒作为刺激物来评估IFNg分泌可能低估了宿主免疫应答。

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 IFNg-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-g 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 percent 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 IFNg secretion may underestimate the host immune response.