LT-IIc, a new member of the type II heat-labile enterotoxin family, exhibits potent immunomodulatory properties that are different from those induced by LT-IIa or LT-IIb

Abstract

A plethora of human pathogens invade and/or colonize mucosal surfaces. Elaboration of strong, protective immune responses against those pathogens by mucosal vaccination, however, is hampered by endogenous regulatory systems in the mucosae that dampen responses to foreign antigens (Ags). To overcome those natural barriers, mucosal adjuvants must be employed. Using a mouse mucosal immunization model and AgI/II, a weak immunogen from Streptococcus mutans, LT-IIc, a new member of the type II subgroup of the heat-labile enterotoxin family, was shown to have potent mucosal adjuvant properties. In comparison to mice intranasally immunized only with AgI/II, co-administration of AgI/II with LT-IIc enhanced production of Ag-specific IgA antibodies in the saliva and vaginal fluids and Ag-specific IgA and IgG in the serum. Secretion of IL-2, IL-6, IL-17, IFN-γ, and TNF-α was enhanced in cultures of AgI/II-stimulated splenic cells isolated from mice that had received LT-IIc as a mucosal adjuvant. In contrast, secretion of IL-10 was suppressed in those cells. This pattern of cytokine secretion suggested that LT-IIc stimulates both Th1 and Th2 immune responses. In contrast to LT-IIa and LT-IIb, the original members of the type II subgroup that also are mucosal adjuvants, LT-IIc dramatically enhanced secretion of IL-1α and IL-1β in peritoneal macrophages that had been co-cultured with LPS. Furthermore, the B pentameric subunit of LT-IIc augmented uptake of Ag by bone marrow-derived dendritic cells to levels that exceeded those attained by use of LPS or by the B pentamers of LT-IIa or LT-IIb. These data confirmed that LT-IIc is a strong mucosal adjuvant with immunomodulatory properties that are distinguishable from those of LT-IIa and LT-IIb and which has immunomodulatory properties that may be exploitable in vaccine development.

Figure 1. Intranasal co-administration of LT-IIc enhances Ag-specific mucosal and systemic immune responses to AgI/II

BALB/c 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-IIc as a mucosal adjuvant. Samples were obtained at day 42 at the time of peak Ag-specific antibody response. Samples were measured for: (A) salivary IgA, (B) vaginal IgA, (C) serum IgA, and (D) serum IgG. Data are reported as arithmetic means; error bars denote one standard error of the mean (n = 5 or 6). Key: statistically different from mice immunized only with AgI/II at P<0.01 (**) and P<0.001 (***).

Figure 2. Production of cytokines by splenic cells

Splenic cells were isolated from intranasally immunized mice at a time point 63 days after the initial immunization. Cultured splenic cells were stimulated in vitro for 4 days with AgI/II (5μg/ml). Culture supernatants from the stimulated splenic cells were measured for: (A) IL-2, (B) IL-6, (C) IFN-γ, (D) TNF-α, (E) IL-17, and (F) IL-10. Data are reported as arithmetic means; error bars denote one standard deviation of the mean (n = 5–6). Key: statistically different from mice immunized only with AgI/II at P<0.05 (*), P<0.01 (**), and P<0.001 (***).

Figure 3. Cytokine production by peritoneal macrophages

Peritoneal macrophages from naïve mice were treated for 4 hrs with 1 μg/ml of LPS prior to addition of LT-IIa, LT-IIb or LT-IIc. After 20 hrs of incubation, the amounts of cytokines in the culture supernatants were determined. Data are reported as arithmetic means; error bars denote one standard error of the mean (n = 3). Key: statistical difference between groups denoted by crossbars at P < 0.01 (**), at P < 0.001 (***); ns, not significantly different between groups denoted by crossbars.

Figure 4. Uptake of FITC-OVA by BMDC is enhanced by LT-IIc-B₅

BMDC were incubated at 37°C with 5 μg/ml of LT-IIa-B₅, 5 μg/ml of LT-IIb-B_5, 5 μg/ml of LT-IIc-B₅, 1 μg/ml of LPS, or PBS (untreated). After 10 min, FITC-OVA (0.2 mg/ml) was added to the cultures. Ten min after addition of FITC-OVA, cells were washed with ice-cold PBS, stained with an APC-conjugated anti-CD11c mAb (BioLegend) and 7-AAD (Calbiochem) and the mean fluorescent intensity (MFI) of CD11c-positive, 7-AAD-negative cells was determined using flow cytometry. Data are reported as arithmetic means; error bars denote one standard error of the mean (n = 3). Data shown represents one of two independent experiments. Fold increases (MFI levels in treated cells/MFI levels in untreated cells) for each group of cells are denoted. Key: statistically different from untreated BMDC at P < 0.005 (**) and P < 0.0001 (***); statistical differences between the values for LT-IIc-B₅ & LT-IIa-B₅ and LT-IIc-B₅ & LT-IIb-B₅ are denoted above the crossbars.