CD1d-dependent B-cell help by NK-like T cells leads to enhanced and sustained production of Bacillus anthracis lethal toxin-neutralizing antibodies

Abstract

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (alpha-galactosylceramide [alpha-GC]) at the time of immunization improves PA-specific Ab responses. We observed that alpha-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d(-/-) mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jalpha281(-/-) mice expressing CD1d but lacking type I alpha-GC-reactive NKT cells, alpha-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

FIG. 1.

CD1d expression and NKT cells are required for optimal production of PA-specific Abs. C57BL/6, CD1d^−/−, and Jα18^−/− mice were immunized s.c. with 10 μg of PA or 10 μg of PA plus 2 μg of α-GC in a 100-μl volume of sterile endotoxin-free PBS. Sera were collected before immunization and on day 28 (data not shown). On day 28 mice were then boosted with half the original dose of PA and sera collected again on day 38 (d38) (a) and day 43 (b). ELISAs were performed to determine the endpoint anti-PA IgG1 titer. Data points represent the endpoint titers for individual mice. (c) Since only two CD1d^−/− mice were used in the first experiment, additional groups of C57BL/6 and CD1d^−/− mice were immunized with PA and analyzed at day 43. (d) Responses in Jα18^−/− mice are shown in a third experiment (also day 43 bleeds). Geometric means are shown. Statistically significant differences between experimental groups are indicated by asterisks.

FIG. 2.

CD1d-dependent NKT activation enhances in vitro lethal toxin neutralization. Sera from experiments with results shown in Fig. 1 were used for in vitro neutralization assays as described in Materials and Methods. Briefly, RAW267.4 macrophages were untreated (none) or incubated with toxin alone or toxin mixed with sera from naïve and immunized mice before assessing viability. Each data point represents the percent cell viability conferred by serum from individual mice. All samples were analyzed in duplicate. Asterisks indicate statistically significant differences between PA-immunized versus PA plus α-GC-immunized groups. (a) Effect of serum dilution on resistance to the toxin. (b) Effect of increasing toxin concentration on neutralization.

FIG. 3.

CD1d-dependent activation of NKT cells enhances in vivo protection against lethal toxin. Naïve and immunized mice were challenged i.v. with PA plus LF at the times indicated by arrows. Survival was then monitored daily for the times indicated. (a) Naïve mice, treated with 0, 50, 200, or 300 μg of each toxin subunit. (b) Survival in C57BL/6 and CD1d^−/− mice, previously immunized with PA and challenged with 200 μg of each toxin subunit. (c) Survival in C57BL/6 previously immunized with PA alone or PA plus α-GC and challenged with the 200-μg dose on day 0 and then the 100-μg dose on day 1. Numbers of mice per group are indicated.

FIG. 4.

α-GC leads to sustained protective anti-PA Ab responses. C57BL/6 mice were immunized with 8 μg of PA alone or PA plus 4 μg of α-GC and then boosted 28 days later with 4 μg of PA alone. Sera were collected at the times indicated before and after booster administration. (a) Endpoint anti-PA IgG1 titers were determined. Data show geometric mean titers (± the 95% confidence interval) for nine mice per group. (b) In vitro neutralization of toxin was assessed at the times shown. Data show the mean percent neutralization for nine mice per group. (c) After 160 days, mice were challenged i.v. with 200 μg of PA and LF on day 0 and 100 μg of PA and LF on day 1 (as indicated by arrows). Survival was then monitored daily. Five mice per group encompassing the range of endpoint anti-PA titers were selected for the challenge experiment. Of the original nine mice, the remaining four mice per group were used for analysis of bone marrow PA-specific plasma cells. However, the sensitivity of the assay was low and the data are not reported here.