Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O

Abstract

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.

FIG. 1

Western blot analysis of LLO in the supernatant of the disrupted liposome used in this study. Liposome (LLO concentration, 30 μg/ml) was disrupted by 0.2% Triton X-100; 100 μl of supernatant fluid was analyzed by SDS-PAGE, and then electrophoresed proteins were transferred to a nitrocellulose sheet. Transferred protein was analyzed by immunoblotting using the anti-LLO monoclonal antibody 7D10E12. Lane A, PBS-liposome; lane B, LLO-liposome.

FIG. 2

Phagocytosis and P-L fusion assay in macrophages. LY-labeled peritoneal exudate macrophages (10⁶/ml) were incubated with liposome for 1 h (B) or without liposome (A). After washing to remove nonphagocytosed liposomes, cells were examined under fluorescence microscopy.

FIG. 3

RT-PCR detection of cytokine-specific mRNA after in vitro stimulation with soluble LLO or LLO-liposome. PEC were unstimulated (lane a) or stimulated with 10 μg of soluble LLO (lane b), PBS-liposome (lane c), or LLO-liposome (lane d) per ml. After stimulation for 60 min, cells were washed to remove nonphagocytosed liposome. Total cellular RNA was extracted for RT-PCR after 6 h of stimulation. Lanes M, 100 bp ladder.

FIG. 4

PCR detection of cytokine-specific mRNA expression after in vivo injection of LLO-liposome. Mice were immunized intravenously with PBS-liposome (lanes a) or LLO-liposome (lanes b). At the time points indicated, total cellular RNA in the spleen was extracted and subjected to RT-PCR. Lanes M, 100 bp ladder.

FIG. 5

Expression of IFN-γ mRNA after immunization with L. monocytogenes ATCC 15313 or EGD as determined by RT-PCR. Mice were immunized with 2 × 10⁷ CFU of ATCC 15313 (lane b), ATCC 15313 plus 200 μl of LLO-liposome (lane c), or 2 × 10³ CFU of EGD (lane d). Lane a, control; lane M, 100 bp ladder. Total cellular RNA was extracted from the spleen 24 h after stimulation for RT-PCR.

FIG. 6

Kinetic change in the CFU of L. monocytogenes EGD and ATCC 15313 after intravenous immunization. Mice were immunized with 10⁴ CFU of EGD (□) or with 2 × 10⁷ CFU of ATCC 15313 alone (•) or with LLO-liposome (200 μl/head) (○). At the time points indicated, CFU counts in the spleen were determined. The hatched area indicates the nondetectable level. Data represent mean counts for three mice ± standard deviation.

FIG. 7

Protective immunity induced by immunization with a combination of 2 × 10⁷ CFU of L. monocytogenes (L. m.) ATCC 15313 and LLO-liposome. The level in mice immunized with virulent strain EGD is shown as positive control. Six days after immunization, mice were challenged intravenously with 2 × 10⁴ CFU of strain EGD, and the CFU number in the spleen was determined 2 days later. The level of protective immunity was calculated as log₁₀ CFU in nonimmune control − log₁₀ CFU in the immune group. Data represent mean counts for three mice ± standard deviation.

FIG. 8

Level of protective immunity conferred by adoptive transfer of T cells. Spleen T cells from each group of immunized mice were transferred into naive recipient mice, and the recipients were challenged intravenously with 2 × 10⁴ CFU of strain EGD. The number of bacteria in the spleen and liver was determined 2 days later. The level of protective immunity was calculated as for Fig. 7. Data represent mean counts for three mice ± standard deviation. **, P < 0.001; *, P < 0.05 compared with the nonimmune control.

FIG. 9

Generation of antigen-specific, IFN-γ-producing cells in mice immunized with a combination of ATCC 15313 and LLO-liposome. Spleen cells (10⁶) obtained from each group of immunized mice were stimulated with 10⁷ HKLM in vitro. IFN-γ titer in the supernatant (A) was determined by ELISA 48 h after stimulation, and ELISPOT assay for the number of antigen (Ag)-specific, IFN-γ-producing T cells (B) was done 18 h after stimulation. Data represent mean counts for three wells ± standard deviation.

FIG. 10

CTL activity in the spleen as determined ex vivo by ⁵¹Cr release assay. Mice were immunized with L. monocytogenes EGD (□), a combination of L. monocytogenes ATCC 15313 and LLO-liposome (○), ATCC 15313 alone (▵), or LLO-liposome alone (•) for 6 days. Cells obtained from nonimmune mice (■) were used as the control. ⁵¹Cr-labeled target cells (BMMΦ) infected with EGD) were cultured with effector cells form each group of mice at ratios ranging form 80:1 to 10:1 for 4 h, and the percent specific lysis was calculated. Data represent mean counts for three wells.