Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants

Abstract

Transcutaneous immunization (TCI) is a new technique that uses the application of vaccine antigens in a solution on the skin to induce potent antibody responses without systemic or local toxicity. We have previously shown that cholera toxin (CT), a potent adjuvant for oral and nasal immunization, can induce both serum and mucosal immunoglobulin G (IgG) and IgA and protect against toxin-mediated mucosal disease when administered by the transcutaneous route. Additionally, CT acts as an adjuvant for coadministered antigens such as tetanus and diphtheria toxoids when applied to the skin. CT, a member of the bacterial ADP-ribosylating exotoxin (bARE) family, is most potent as an adjuvant when the A-B subunits are present and functional. We now show that TCI induces secondary antibody responses to coadministered antigens as well as to CT in response to boosting immunizations. IgG antibodies to coadministered antigens were also found in the stools and lung washes of immunized mice, suggesting that TCI may target mucosal pathogens. Mice immunized by the transcutaneous route with tetanus fragment C and CT developed anti-tetanus toxoid antibodies and were protected against systemic tetanus toxin challenge. We also show that bAREs, similarly organized as A-B subunits, as well as the B subunit of CT alone, induced antibody responses to themselves when given via TCI. Thus, TCI appears to induce potent, protective immune responses to both systemic and mucosal challenge and offers significant potential practical advantages for vaccine delivery.

FIG. 1

Dose-dependent antibody response to CT (A) or LT (B) in mice immunized by the transcutaneous route. (A) The mice (n = 5) were immunized at 0, 4, and 8 weeks, and serum was analyzed for anti-CT IgG (H+L) titers at 12 weeks. (B) The mice (n = 5) were immunized at 0 weeks, and serum was analyzed for anti-LT IgG (H+L) titers 3 weeks later. The results shown are the geometric mean and SEM of IgG titers measured in sera from five individual animals and reported in ELISA units, the inverse dilution at which the absorbency is equal to 1.0 at 405 nm. An asterisk denotes a statistically significant difference (P ≤ 0.05) in antibody titer compared to the response in the high-dose groups.

FIG. 2

Kinetics of the IgG (H+L) antibody response to CT (A, C, E), LT (B), DT (D), or BSA (F) in animals immunized and boosted (arrows) by the transcutaneous route. The mice (n = 5) were immunized with CT alone (100 μg) (A), LT alone (100 μg) (B), CT plus DT (100 μg of CT + 100 μg of DT [black circles]) (C and D), DT alone (100 μg [gray circles]) (D), CT plus BSA (100 μg of CT + 200 μg of BSA [black circles]) (E and F), or BSA alone (200 μg [gray circles]) (F). Gray circles in panels D and F indicate antibody levels to DT or BSA, respectively, in mice vaccinated without using CT as the adjuvant. Antibody titers were measured by ELISA at multiple time points. The results are reported as the mean ± SEM. Similar results were obtained in two independent experiments.

FIG. 3

TCI of mice with preexisting anti-CT and anti-DT titers with a third, unrelated antigen (BSA). The mice were immunized by the transcutaneous route at 0, 4, and 8 weeks with CT (100 μg) and DT (100 μg) and then exposed to CT (100 μg) and BSA (200 μg) at 13 and 17 weeks after the first immunization. Serum collected from the animals at 0, 13, and 20 weeks was analyzed. The results are reported as the mean ± SEM for groups of five mice. Similar results were obtained with BSA as the primary immunogen and DT as the second immunogen.

FIG. 4

Responses to CT and coadministered antigens DT at the mucosa. Mice (n = 5) were immunized with CT (100 μg) (left), DT (100 μg) (middle), or CT plus DT (100 μg each) (right) at 0, 8, and 18 weeks by TCI. At 30 weeks later, stool samples and lung wash extracts were collected and analyzed for anti-CT (A and B) and anti-DT (C) Ig. Antigen-specific IgG (A and C) and IgA (B) titers were measured. Curves showing the titers from individual animals are plotted. Control stool preimmune samples from mice and control lung washes from mice immunized with an irrelevant protein, ricin A subunit, had optical densities of 0. The results are representative of data observed in two independent experiments.

FIG. 4

Responses to CT and coadministered antigens DT at the mucosa. Mice (n = 5) were immunized with CT (100 μg) (left), DT (100 μg) (middle), or CT plus DT (100 μg each) (right) at 0, 8, and 18 weeks by TCI. At 30 weeks later, stool samples and lung wash extracts were collected and analyzed for anti-CT (A and B) and anti-DT (C) Ig. Antigen-specific IgG (A and C) and IgA (B) titers were measured. Curves showing the titers from individual animals are plotted. Control stool preimmune samples from mice and control lung washes from mice immunized with an irrelevant protein, ricin A subunit, had optical densities of 0. The results are representative of data observed in two independent experiments.