ANALYSIS OF IMMUNOREGULATORY VACCINIA GENES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。天花病毒和牛痘病毒含有大量的免疫调节基因产物，这些基因产物被认为通过调节宿主的免疫反应来增强病毒的繁殖。干扰素γ (IFNgamma)的分泌是体外细胞免疫反应的常用测量方法。我们假设牛痘病毒B8R蛋白（一种分泌的IFNgamma受体类似物）可能干扰ELISPOT法检测IFNgamma。采用ELISPOT法测定外周血单个核细胞（PBMCs）在牛痘病毒或eb病毒转化的自体B细胞刺激下ifnγ的分泌量。牛痘感染抑制CD8+ T细胞对自体ebv转化的B细胞株的ifn - γ ELISPOT反应70%。相比之下，ebv特异性ifn - γ ELISPOT反应不受修饰安卡拉牛痘（MVA）感染的抑制，MVA是一种不含B8R的减毒牛痘菌株。用B8R基因缺失的痘苗病毒重组物刺激PBMCs，与野生型菌株相比，IFNgamma分泌的检测增加了75%。与野生型菌株相比，MVA刺激牛痘疫苗接种者的PBMCs导致IFNgamma分泌增加12倍。牛痘病毒免疫调节基因产物干扰细胞免疫反应的体外检测，包括牛痘特异性和ebv特异性反应。牛痘病毒中可溶性ifn - γ受体（B8R）的缺失导致这种作用不完全消除，这表明MVA中缺失的其他基因也有助于逃避CD8+ T细胞介导的免疫反应。这些结果表明，使用牛痘病毒作为刺激来评估IFNgamma分泌可能低估了宿主的免疫反应

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