Binding to gangliosides containing N-acetylneuraminic acid is sufficient to mediate the immunomodulatory properties of the nontoxic mucosal adjuvant LT-IIb(T13I)

Abstract

By use of a mouse mucosal immunization model, LT-IIb(T13I), a nontoxic mutant type II heat-labile enterotoxin, was shown to have potent mucosal and systemic adjuvant properties. In contrast to LT-IIb, which binds strongly to ganglioside receptors decorated with either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), LT-IIb(T13I) binds NeuAc gangliosides much less well. Rather, LT-IIb(T13I) binds preferentially to NeuGc gangliosides. To determine if the adjuvant properties of LT-IIb(T13I) are altered in the absence of NeuGc ganglioside receptors, experiments were conducted using a Cmah-null mouse line which is deficient in the synthesis of NeuGc gangliosides. Several immunomodulatory properties of LT-IIb(T13I) were shown to be dependent on NeuGc gangliosides. LT-IIb(T13I) had reduced binding activity for NeuGc-deficient B cells and macrophages; binding to NeuGc-deficient T cells and dendritic cells (DC) was essentially undetectable. Treatment of Cmah-null macrophages with LT-IIb(T13I), however, upregulated the transcription of interleukin-4 (IL-4), IL-6, IL-17, and gamma interferon (IFN-gamma), four cytokines important for promoting immune responses. The production of mucosal IgA and serum IgG against an immunizing antigen was augmented in NeuGc-deficient mice administered LT-IIb(T13I) as a mucosal adjuvant. Notably, NeuGc gangliosides are not expressed in humans. Still, treatment of human monocytes with LT-IIb(T13I) induced the secretion of IL-6, an inflammatory cytokine that mediates differential control of leukocyte activation. These results suggested that NeuAc gangliosides are sufficient to mediate the immunomodulatory properties of LT-IIb(T13I) in mice and in human cells. The nontoxic mutant enterotoxin LT-IIb(T13I), therefore, is potentially a new and safe human mucosal adjuvant.

FIG. 1.

Patterns of binding of LT-IIb and LT-IIb(T13I) to splenic lymphoid cells isolated from C57BL/6J or Cmah-null mice. Histograms were gated on CD4⁺ (helper T cells), CD8⁺ (cytotoxic T cells), B220⁺ (B cells), CD11b⁺ (macrophages), or CD11c⁺ (DC) cells. Dead cells were excluded by staining with 7-AAD. Shaded histograms, control cells (nonspecific binding of antibodies in the absence of enterotoxin); thin-line histograms, LT-IIb; bold-line histograms, LT-IIb(T13I). A shift of the fluorescence intensity to the left indicates a decrease in or absence of binding of enterotoxin to the cells. Histograms are derived from data obtained from one of three independent replicate experiments. APC, allophycocyanin.

FIG. 2.

Two-dimensional thin-layer chromatograms for gangliosides isolated from macrophages obtained from thioglycolate-treated C57BL/6J mice (B) or homozygous Cmah-null mice (C) and the corresponding schematic diagram (A). (A) Solid ellipses indicate NeuGc gangliosides; shaded ellipses indicate mixed NeuGc- and NeuAc-containing gangliosides; open ellipses indicate NeuAc gangliosides. Numbers in the schematic diagram (reprinted from reference with permission of the publisher) correspond to structures as follows: 1α, GD1α-NeuGc; 2α, GD1α-NeuAc/NeuGc; 3α, GD1α-NeuAc; 1, GD1a-NeuGc; 2, GDla-NeuAc/NeuGc; 3, GD1a-NeuAc; 4, GM1b-NeuGc; 5, GM1a-NeuGc; 6, GM1b-NeuAc; 7, GM1a-NeuAc; 8, GM2-NeuGc; 9, GM2-NeuAc; 10, GM3-NeuGc; 11, GM3-NeuAc. (B and C) Ellipses denote NeuGc gangliosides expressed in C57BL/6J mice (B) or the positions of the missing NeuGc gangliosides in Cmah-null mice (C). Arrows and numbers in the lower right corner indicate the directions of movement of the first and second solvents. The origin is denoted by a dot in the lower right corner. Ganglioside standards, resolved in each dimension, are noted along the top (solvent 1) and left (solvent 2) of the chromatographs as follows: a, GM3; b, GM2; c, GM1; d, GD3; e, GD1a; f, GD1b.

FIG. 3.

Effects of LT-IIb and LT-IIb(T13I) on salivary IgA (A), vaginal IgA (B), and serum IgG (C) antibody responses to AgI/II. C57BL/6J and Cmah-null mice were immunized intranasally on days 1, 14, and 28 with 10 μg of AgI/II in the presence or absence of 1 μg of LT-IIb or LT-IIb(T13I) as a mucosal adjuvant. Samples were collected 2 weeks after the second booster immunization at the time of peak Ag-specific antibody response. The data are arithmetic means ± standard errors of the means (n, 5 to 6). Statistical differences from the group immunized only with AgI/II are noted as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG. 4.

Effects of LT-IIb and LT-IIb(T13I) on human monocytes. (A) Binding of LT-IIb and LT-IIb(T13I) to human monocytes. (B) Induction of IL-6 by human monocytes after treatment with LT-IIb or LT-IIb(T13I). (C) Accumulation of cAMP in human monocytes treated with LT-IIb, LT-IIb(T13I), or LT-IIb-B₅. Fold increases (cAMP levels in treated cells/cAMP levels in untreated cells) for each set are given above the bars. The data are arithmetic means ± standard errors of the means (n = 3). Double asterisks indicate statistical differences from the LT-IIb group (P < 0.01).