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

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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.