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Determination of the critical region of listeriolysin O (LLO) for the cytokine-inducing activity and application of LLO to vaccination with killed BCG.

确定李斯特菌溶血素 O (LLO) 细胞因子诱导活性的关键区域以及 LLO 在灭活卡介苗疫苗接种中的应用。

基本信息

批准号：
11670263
负责人：
KAWAMURA Ikuo
金额：
$ 2.43万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Listeriolysin O (LLO) is demonstrated to be a mojor virulence factor of Listeria monocytogenes and is one of the member of cholesterol-binding cytolysins. we have reported that LLO induces endogenous Th1-type cytokine productions independent of the membrane lytic activity. It suggests that LLO may play a role as an adjuvant to promote the generation of protective immunity if it does not exert the cytolytic activity in vivo. In the present study, therefore, we attempted to determine the critical region of LLO only for the cytokine-inducing activity and evaluated its effectiveness as an adjuvant by administration of LLO encapsulated with liposome containing cholesterol.LLO deleted for the fourth domain (LLO415), which is important for expression of the cytolytic activity, showed IFN-γ inducing activity. However, recombinant fourth domain did not induce the cytokine production. The activity of LLO415 was considerably suppressed when the N-terminal portion was further deleted. These data indicated that the N-terminus of LLO but not C-terminal fourth domain is critical for the cytokine-inducing activity. The activity of LLO was not observed in spleen cells of C3H/HeJ mice and was blocked by anti-CD14 antibody, suggesting that the signal of LLO may transduce via a signaling pathway of LPS.Moreover, immunoprecipitation revealed that LLO bound to two molecules on J774.1 cells, of which molecular weights are approximately 50-60kDa. We believe that these molecules are involved in the signal transduction of LLO.To determine the efficacy of LLO as an adjuvant, C3H/HeN mice were immunized with killed BCG along with LLO encapsulated in the liposome. The LLO showed no cytolytic activity and promoted generation of protective T cells, suggesting that LLO functions as the adjuvant to promote Th1-type host resistance. On the other hand, because LLO415 was not trapped in liposome well, the adjuvant activity was remained to be elucidated.

李斯特菌溶血素O （Listeriolysin O， LLO）是单核增生李斯特菌的主要毒力因子，是胆固醇结合溶细胞素的成员之一。我们已经报道了LLO诱导内源性th1型细胞因子的产生，而不依赖于膜裂解活性。提示LLO在体内不发挥细胞溶解作用的情况下，可能起到促进保护性免疫产生的佐剂作用。因此，在本研究中，我们试图确定LLO仅具有细胞因子诱导活性的关键区域，并通过使用含有胆固醇的脂质体包裹的LLO来评估其作为佐剂的有效性。LLO缺失的第四个结构域（LLO415）对细胞溶解活性的表达很重要，显示出IFN-γ诱导活性。然而，重组第四结构域不能诱导细胞因子的产生。当n端被进一步删除时，LLO415的活性被显著抑制。这些数据表明LLO的n端而不是c端第四结构域对细胞因子诱导活性起关键作用。在C3H/HeJ小鼠脾细胞中未观察到LLO的活性，并被抗cd14抗体阻断，提示LLO信号可能通过LPS信号通路转导。此外，免疫沉淀显示LLO在J774.1细胞上结合了两个分子量约为50-60kDa的分子。我们认为这些分子参与了LLO的信号转导。为了确定LLO作为佐剂的作用，我们将杀死的BCG与包封在脂质体中的LLO一起免疫C3H/HeN小鼠。LLO没有细胞溶解活性，并促进保护性T细胞的产生，提示LLO作为佐剂促进th1型宿主的抗性。另一方面，由于LLO415在脂质体中未被很好地捕获，其佐剂活性尚待阐明。