Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants

Abstract

Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i. n. immunizations were consistently higher than the equivalent p.o. -delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response.

FIG. 1

Sensitivity of LT, LT-K63, LT-G192, and LT-K63/G192 protease activity. The structural subunits of LT are highlighted. The smallest protein is the monomeric form of the B subunit (B_m). The remaining two proteins are the uncleaved enzymatically active A subunit and the proteolytically cleaved A₁ subunit. (A) Proteolytic breakdown products after treatment of the proteins with trypsin for 90 min under nondenaturing conditions; (B) breakdown pattern observed after incubation with trypsin for 5 min under denaturing conditions; (C) breakdown pattern observed after incubation with trypsin for 15 min under denaturing conditions; (D) proteolytic breakdown pattern of the proteins after incubation with protease-rich intestinal lavage fluid.

FIG. 2

Serum and local antibody responses to KLH in i.n.-immunized mice. (a) Total anti-KLH response in sera of mice on days 0 (□), 20 (), 34 (■), and 49 (). The results are mean titers calculated from individual animals. Error bars represent the standard deviation of the mean response. (b) Variation in individual titers of KLH specific antibody in the sera of mice at a single time point on day 49. (c) Mean IgA response detected in the lung lavage samples from these mice on day 49. Bar represent mean titers for each group.

FIG. 3

Serum and local antibody responses to KLH in p.o.-immunized mice. (a) Mean antibody response of p.o.-immunized mice on days 0 (□), 20 (), 34 (■), and 49 (). The results are mean titers calculated from individual animals. Error bars represent standard deviations of the mean responses. (b) Variation in individual titers of KLH-specific antibody in sera of mice on day 49. (c) Titers of KLH-specific IgA from intestinal washes from individual animals on day 49. Limited intestinal samples were available for testing from groups immunized with LT-G192 or LT-K63/G192.

FIG. 4

T-cell proliferative responses to KLH in vitro. Splenocytes from individual mice were isolated and stimulated with various concentrations of KLH. Each bar represents the mean stimulation index for a group of mice at the optimal antigen stimulating concentration of 10 μg/ml; Error bars represent standard deviations of the means.

FIG. 5

IgG subclass analysis of the anti-KLH response in sera taken on day 49 of the experiment from p.o.-immunized mice. Mean antibody titers of KLH-specific IgG1 (■), IgG2a (), and IgG2b (▨) were calculated from at least eight mice per group. Error bars represent standard deviations of the mean responses.