Department of Veterinary Biosciences, Ohio State University, Columbus, OH 43210, USA.
J Immunol. 2010 Nov 15;185(10):5943-52. doi: 10.4049/jimmunol.0902795. Epub 2010 Oct 15.
We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.
我们已经表明，将炭疽芽孢杆菌保护性抗原（PA）与一种具有降低腺嘌呤环化酶活性的炭疽杆菌水肿因子（EF）突变体（即 EF-S414N）鼻内共应用增强了抗 PA Ab 反应，但也作为粘膜佐剂共同给予了无关的 Ag。为了阐明水肿毒素（EdTx）成分在其佐剂活性中的作用，我们研究了缺乏与 EF 结合能力的 PA 突变体（PA-U7）或另一种突变体（PA-dFF）如何影响 EdTx 诱导的适应性免疫，该突变体允许 EF 的细胞摄取，但不能有效地将其易位到细胞质中。天然 EdTx 通过巨噬细胞和 B 淋巴细胞促进共刺激分子表达，并通过体外宫颈淋巴结细胞产生广泛的细胞因子反应。当用 EF 加 PA-dFF 或 PA-U7 代替 PA 处理细胞时，这些作用会降低或消除。我们还通过与 EdTx 变体（野生型或突变型 PA 和 EF）一起用鼠疫耶尔森氏菌 F1 和 LcrV Ag 的重组融合蛋白（F1-V）鼻内免疫了几组小鼠。对针对 F1-V 或 EdTx 成分（即 PA 和 EF）的血清和粘膜 Ab 反应的分析表明，接受 PA-U7 代替 PA 的小鼠没有佐剂活性。相比之下，用 PA-dFF 共免疫增强了血清 Ab 反应。最后，用天然 PA 和缺乏腺嘌呤环化酶活性的 EF 突变体（EF-K346R）免疫未能增强 Ab 反应。总之，EdTx 作为粘膜佐剂需要完全功能的 PA 和至少腺嘌呤环化酶活性。
