Epicutaneous immunotherapy on intact skin using a new delivery system in a murine model of allergy (1)
Viaskin is a new epicutaneous delivery system (EDS) that, without any skin preparation or adjuvant, promotes diffu- sion of allergens in the thickness of the stratum corneum and towards the immune cells of the epidermis.
Immunization protocol: Mice were sensitized to OVA (n = 18), pollen (n = 18) or HDM (n = 24) by means of two subcutaneous injections at 1 week interval on the back of the neck with: 10 ug of OVA (grade V, ), 10 ug of D. glomerata pollen using a protocol adapted from Wiedermann et al. or 10 ug of Dermato- phagoides farinae. These three allergen solutions were added to 1.6 ug of aluminium hydroxide. One week after the second sensitization, the mice were boosted by an intranasal administration of 10 ug of OVA, pollen or HDM in 50 uL of 0.9% NaCl. Mice were sensitized to peanut (n = 18) by means of a total of six intra-gastric gavages every 6 days with 1ug of homogenized peanut protein extract mixed with 10 ug of cholera toxin. One week after the last gavages, the mice were boosted by an intranasal administration of 10mg of peanut in 50 uL of 0.9% NaCl. Sensitization to allergens was monitored in the blood samples by the production of specific Ig (sIg)E, 10 days after the last injection. Matching groups of control mice (n = 6 for OVA, pollen and peanut, n=8 for HDM) received either 50uL of 0.9% NaCl by subcutaneous administrations or 200 uL of phosphate-buffered saline by oral administrations using the same scheme as for the sensitized mice.
Treatment: Viaskins is composed of a central translucent polyethylene membrane (11 mm in diameter) surrounded by an adhesive polyester non-woven crown as contact support to maintain the chamber on the skin. A dry layer of allergenic materials, containing OVA, pollen, HDM or peanut proteins was deposited on this backing (100 ug total protein of each allergen). The delivery system creates an occlusive chamber on the skin that rapidly generates moisture and releases the allergens from the plastic support to the skin, allowing adequate diffusion of the proteins towards the epidermal immune-competent cells. Mice were anaesthetized intraperitoneally with 100 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine and shaved using an electric clipper and a depilatory cream without corticoid. Twenty-four hours later, after total recovery of the skin, mice were anaesthetized and EDS, with 100 mg OVA, pollen, HDM or peanut proteins, were placed on the back of the mice and maintained using a bandage for 48 h. As hair grows fast in mice, the skin was prepared before each application as described above.
In EPIT groups, IL-4, IL-5, IL-13 and eotaxin decreased significantly in the BAL fluid with all allergens. Similar results were observed in the serum. SCIT groups showed similar decreases in BAL and serum cytokines, except for pollen-sensitized mice in which the cytokine levels were not different from NT groups. Because of the low level of this cytokine, no difference could be detected between groups in mice sensitized to HDM or peanut allergens. IL-10 and IL-17 were detected in the BAL fluid and sera without any significant difference between the groups. A slight decrease of TNF-a was measured in the BAL fluid between treated groups (EPIT and SCIT) and NT groups in HDM and peanut experiments.
At the end of the treatment (D60), sIg levels evolved differently in the treated groups of each allergen experiment but without any significant difference between SCIT and EPIT. For OVA, sIgE decreased in SCIT and EPIT groups and the values were significantly lower when compared with NT (P<0.05). For peanut, whereas sIgG1 remained unchanged, the treatment signifi- cantly decreased sIgE (P<0.05 as compared with NT) and increased sIgG2a (P<0.001). For pollen, sIgE, sIgG1 and sIgG2a increased (P<0.05) in the treated groups. For HDM, while sIgE level did not vary, sIgG1 and sIgG2a increased (P<0.05 to 0.001). As a result, with SC and EP treatments, the IgG1/IgG2a ratio decreased significantly in OVA and peanut experiments, remained identical with pollen and increased with HDM.
Recently, a new protocol of sensitization to OVA in BALB/c mice without an adjuvant has been proposed. Comparable values of sIgE were obtained in the present study but higher values of IgG1 were observed. During immunotherapy, OVA-specific IgE, IgG1 and IgG2a increased until the fifth week of treatment. Then, IgE exhib- ited a sharp decrease whereas IgG2a continued to increase and OVA-specific IgG1 also increased for two additional weeks before declining slighter than that of sIgE. The administration of purified Ara h 2 or a mixture of purified Ara h 1 + Ara h 2 did not exhibit better serological response than the use of the full natural extract used in the present study. Fluorochrome-conjugated OVA that, after 24h of EDS application, fluorescence detected in the superficial layers of the skin was only localized in the antigen-presenting cells. More than 90% of the Langherans cells of the epidermis and almost 50% of the dendritic cells of the dermis under the EDS application area trapped the allergen within the first 24 h of application.