Comparative analysis of the mucosal adjuvanticity of the type II heat-labile enterotoxins LT-IIa and LT-IIb

Abstract

Cholera toxin (CT) and the heat-labile enterotoxin of Escherichia coli (LT-I) are members of the serogroup I heat-labile enterotoxins (HLT) and can serve as systemic and mucosal adjuvants. However, information is lacking with respect to the structurally related but antigenically distinct serogroup II HLT, LT-IIa and LT-IIb, which have different binding specificities for ganglioside receptors. The purpose of this study was to assess the effectiveness of LT-IIa and LT-IIb as mucosal adjuvants in comparison to the prototypical type I HLT, CT. BALB/c mice were immunized by the intranasal (i.n.) route with the surface protein adhesin AgI/II of Streptococcus mutans alone or supplemented with an adjuvant amount of CT, LT-IIa, or LT-IIb. Antigen-specific antibody responses in saliva, vaginal wash, and plasma were assayed by enzyme-linked immunosorbent assay. Mice given AgI/II with LT-IIa or LT-IIb by the i.n. route had significantly higher mucosal and systemic antibody responses than mice immunized with AgI/II alone. Anti-AgI/II immunoglobulin A (IgA) antibody activity in saliva and vaginal secretions of mice given AgI/II with LT-IIa or LT-IIb was statistically similar in magnitude to that seen in mice given AgI/II and CT. LT-IIb significantly enhanced the number of AgI/II-specific antibody-secreting cells in the draining superficial cervical lymph nodes compared to LT-IIa and CT. LT-IIb and CT induced significantly higher plasma anti-AgI/II IgG titers compared to LT-IIa. When LT-IIb was used as adjuvant, the proportion of plasma IgG2a relative to IgG1 anti-AgI/II antibody was elevated in contrast to the predominance of IgG1 antibodies promoted by AgI/II alone or when CT or LT-IIa was used. In vitro stimulation of AgI/II-specific cells from the superficial lymph nodes and spleen revealed that LT-IIa and LT-IIb induced secretion of interleukin-4 and significantly higher levels of gamma interferon compared to CT. These results demonstrate that the type II HLT LT-IIa and LT-IIb exhibit potent and distinct adjuvant properties for stimulating immune responses to a noncoupled protein immunogen after mucosal immunization.

FIG. 1

(A) SDS-PAGE of purified LT-IIa (lane 1) and LT-IIb (lane 2) dissociated into the A subunit (∼28 kDa) and B-subunit monomers (∼12.5 kDa). (B) Western blot of recombinant LT-IIa (lane 1) and LT-IIb (lane 2) probed with polyclonal antibodies to LT-IIa and LT-IIb, respectively. Numbers at the left indicate molecular masses in kilodaltons.

FIG. 2

Salivary IgA (A) and vaginal IgA (B) and IgG (C) antibody responses to AgI/II after i.n. immunization of mice with AgI/II alone or with CT, LT-IIa, or LT-IIb as adjuvant. Results are the arithmetic means ± standard deviations for seven mice. Asterisks in panel C indicate statistically significant differences at P < 0.05 compared to LT-IIa.

FIG. 3

Plasma IgG (A), IgA (B), and IgG subclass (C) antibody responses to AgI/II from mice immunized i.n. with AgI/II alone or with CT, LT-IIa, or LT-IIb. Data represent the arithmetic means ± standard deviations for seven mice. In panel A, ∗, ∗∗, and ∗∗∗ indicate statistically significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively, compared to LT-IIa. In panel B, ∗ and ∗∗ indicate significant differences at P < 0.05 and P < 0.01, respectively, compared to CT and LT-IIa. In panel C, ∗∗∗ indicates significant difference (P < 0.001) compared to LT-IIa and CT and ∗ indicates significant difference (P < 0.05) compared to LT-IIa and LT-IIb.

FIG. 4

Proliferative responses of cells from the central posterior lymph nodes (A) and spleen (B) cultured in vitro with AgI/II for 4 days. Central posterior lymph nodes and spleen were excised 40 days after the last immunization. Results are shown as the stimulation index determined by [³H]thymidine incorporation. The data presented are the mean stimulation index ± standard deviation of quadruplicate cultures. In A, ∗ and ∗∗ indicate statistical differences at P < 0.05 and P < 0.01, respectively, compared to LT-IIa and CT. Background [³H]thymidine incorporation in unstimulated cultures was as follows: for central posterior lymph nodes, AgI/II, 698 ± 136; AgI/II + CT, 3,121 ± 1,402; AgI/II + LT-IIa, 4,166 ± 1,678; and AgI/II + LT-IIb, 3,222 ± 216; for spleen, AgI/II, 1,687 ± 436; AgI/II + CT, 2,849 ± 786; AgI/II + LT-IIa, 2,249 ± 317; and AgI/II + LT-IIb, 2,703 ± 511.

FIG. 5

Production of IFN-γ and IL-4 by AgI/II-specific cells from superficial lymph nodes (A) and spleens (B and C) of BALB/c mice immunized i.n. with AgI/II with or without CT, LT-IIa, or LT-IIb. Cells were stimulated in vitro with AgI/II at the concentrations shown for 4 days. Data shown are the arithmetic mean values ± standard deviations (n = 4) as determined by cytokine-specific ELISA. In panels A and B, ∗, ∗∗, and ∗∗∗ indicate significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively, compared to CT. In panel C, ∗, ∗∗, and ∗∗∗ indicate significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively, compared to LT-IIa and LT-IIb.

FIG. 6

Comparison of anti-AgI/II ASC from the NALT (A), spleens (B), and superficial lymph nodes (C) of mice immunized i.n. with AgI/II with or without CT, LT-IIa, or LT-IIb as adjuvant. Data shown are the mean numbers of anti-AgI/II ASC per 10⁶ cells ± standard deviations (n = 3). In panel A, ∗ indicates statistical significance at P < 0.05 compared to LT-IIa; In panel C, ∗∗∗ indicates significant difference at P < 0.001 compared to CT and LT-IIa and ∗ indicates statistical significance at P < 0.05 compared to LT-IIa.