A second generation of double mutant cholera toxin adjuvants: Enhanced immunity without intracellular trafficking
A second generation of double mutant cholera toxin adjuvants: Enhanced immunity without intracellular trafficking
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DOI：
10.4049/jimmunol.177.5.3045
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发表时间：
2006-09-01
影响因子：
4.4
通讯作者：
Fujihashi, Kohtaro
中科院分区：
文献类型：
--
作者：
Hagiwara, Yukari;Kawamura, Yuki I.;Fujihashi, Kohtaro
鼻内应用天然霍乱毒素（nCT）作为粘膜佐剂，通过与嗅觉神经中的GM 1神经节苷脂结合，对中枢神经系统具有潜在毒性。尽管已经开发出了显示粘膜佐剂活性而无毒性的霍乱毒素（mCTs）突变体，但仍然不清楚这些mCTs是否会诱导CNS损伤。为了帮助克服这些问题，在这项研究中：我们创造了新的双突变CT（dmCT），在ADP-核糖基转移酶活性中心（E112 K）和COOH-末端KDEL（E112 K/KDEV或E112 K/KDGL）中有两个氨基酸取代。共聚焦显微镜分析表明，在肠上皮T84细胞中，dmCTs的细胞内定位不同于mCTs和nCTs。此外，两种dmCT在Y1细胞试验和小鼠回肠袢试验中均显示出极低的毒性。当检查粘膜佐剂性时，两种dmCT在粘膜和全身淋巴组织中均诱导增强的OVA特异性免疫应答。有趣的是，尽管dmCT E112 K/KDEV和dmCT E112 K/KDGL均显示出OVA特异性CD 4（+）T细胞的高Th 2型和显著的Th 1型细胞因子应答，但dmCT E112 K/KDEV表现出显著低于nCT和dmCT E112 K/KDGL的Th 1型细胞因子应答。这些结果表明，新开发的dmCT保留了强的生物佐剂活性，而没有CNS毒性。
Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study: we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Thl-type cytokine responses by OVA-specific CD4(+) T cells, dmCT E112K/KDEV exhibited significantly lower Thl-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.