CT is responsible for the symptoms of cholera1, is produced by the bacterium Vibrio cholerae and is a member of the adenosine diphosphate (ADP)-ribosylating bacterial exotoxins2. CT is already widely used experimentally as an adjuvant3, a compound used to enhance the immune response to vaccine components. Given by the oral or nasal route with proteins4 or vaccine components such as tetanus toxoid5, CT and its derivatives enhance the resulting antibody responses.

Transcutaneous immunization (immunization using the skin) is achieved by simple application of a solution to the surface of the skin. To gain access to mouse skin, we shaved the animals' backs with a clipper and allowed them to rest for 24 hours before immunization. To avoid the possibility of oral immunization occurring through grooming, we anaesthetized the mice and then wetted the skin surface with immunizing solution for 120 minutes. Finally, we washed the skin extensively with water. No redness or swelling was observed at the immunization site for up to 72 h after the primary or boosting immunizations, and skin biopsies at the site of exposure showed no signs of inflammation. In separate experiments to exclude the possibility of immunization secondary to skin disruption, we also immunized mice on an unshaved ear.

Transcutaneous immunization was first achieved using CT by itself. When given alone by the oral route, CT stimulates a potent immune response in the form of anti-CT antibodies6. We tested the capacity of CT to induce a similar response through the skin by applying a saline solution of CT to the bare skin of the shaved mouse. This application induced high levels of IgG antibodies specific for CT (Fig. 1a).

Figure 1: Individual IgG antibody responses to antigens using cholera toxin (CT) as adjuvant.
figure 1

BALB/c mice (n=5) were immunized transcutaneously with a saline solution of: a, 100 μg CT; b, 200 μg of bovine serum albumin (BSA), or 200 μg of BSA and 100 μg of CT; c, 100 μg of diphtheria toxoid (DT) or 100 μg of DT and 100 μg of CT; d, 50 μg of tetanus toxoid (TT) or 50 μg of TT and 100 μg of CT. Mice were immunized and boosted at 8 weeks (a, c, d) or 3 weeks (b) later. Sera were analysed for IgG antibodies by solid-phase ELISA two weeks after the second immunization and compared to pre-immune sera. Antigen-specific IgG responses were determined and are reported as the geometric mean of individual sera in ELISA units, the inverse dilution at which the absorbance (405 nm) is equal to 1.0.

The high levels of antibodies produced against CT administered by the transcutaneous route suggested that CT might also act as a skin adjuvant to enhance the immune response to proteins and vaccine components placed on the skin.

Bovine serum albumin (BSA) is a large protein antigen of the type that, when given by the mucosal route, requires an adjuvant such as CT to induce an immune response3. We achieved induction of BSA-specific antibodies only when BSA was coadministered with CT (Fig. 1b). Thus, CT acted as a transcutaneous adjuvant, promoting the immune response to BSA. CT also acted as a transcutaneous adjuvant for the common vaccine components diphtheria toxoid and tetanus toxoid (Fig. 1c, d).

A critical role in enhancing the immune response to both licensed and experimental vaccinesis played by adjuvants7. These compounds, and especially CT and related products, have been important in the development of the mucosal route as a useful and easily accessible non-invasive way to induce immunity3. Employing CT as an adjuvant for transcutaneous immunization would allow the skin to be used in a similar way. This method could be particularly useful given the large surface area of the skin, its accessibility and the existence of potent immune cells within it8.

Skin immune responses can also be induced by skin disruption with depilatory agents9 or by scarification as in smallpox inoculation10, but transcutaneous immunization involving CT offers the benefit of leaving the skin intact. Using CT to induce immune responses through the skin to common off-the-shelf vaccine components — such as tetanus toxoid and diphtheria toxoid — may ultimately lead to the development of simple and safe needle-free vaccines.