Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Abstract

Background: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods: The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results: Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

Keywords: Viaskin; anaphylaxis mouse model; cashew allergy; epicutaneous immunotherapy.

Figure 1

Development and characterization of mouse model of cashew sensitization and anaphylaxis. Mice were sensitized to cashew through skin (in red) or oral (in green) routes. As a negative control, a group of naïve mice (in white) was included. (A) Cashew‐specific IgE, IgG1, and IgG2a antibody responses were evaluated by ELISA, from blood samples collected one week after the end of the sensitization. Mice were orally challenged to cashew one week after the end of the sensitization. (B) Body temperature was measured every 5 minutes following challenge for 60 minutes. (C) Area under the curve was calculated for each individual percentage of temperature variation curve using 100% as a baseline. (D) Diarrhea occurrence and severity was scored based on Table S2. mMCP‐1 (E) and mMCP‐7 (F) concentrations were measured by ELISA from plasma collected immediately after the challenge, (n = 8 per experimental group). Data are median with interquartile range of individual values. P values were determined using the Mann‐Whitney unpaired t test (**P < 0,01; ***P < 0,001; n.s., nonsignificant). For A, C, E, and F panels, the level of significant measured between each sensitized group and the negative control group is indicated above each graph

Figure 2

Validation of the oral route of sensitization as a trigger of IgE‐mediated anaphylaxis in mice. Mice were orally sensitized to cashew. One week after the end of the sensitization, mice received IgE blocking antibody (clone EM‐95, in green), IgG blocking antibody (anti‐FcγRII/RIII clone 2.4G2, in blue) or relevant isotype controls (in red). The day after, mice were challenged orally to cashew. (A) Body temperature was measured every 5 minutes following challenge for 60 minutes and (B) diarrhea occurrence and severity was scored based on Table S2. mMCP‐1 (C) and mMCP‐7 (D) concentrations were measured by ELISA from plasma collected immediately after the challenge, (n = 8 per experimental group). Data are median with interquartile range of individual values. P values were determined using the Mann‐Whitney unpaired t test (*P<.05; ***P<.001; n.s., nonsignificant). For C and D panels, the level of significant measured between each sensitized group and the negative control group is indicated above each dot plot

Figure 3

Cashew allergens are efficaciously delivered by epicutaneous patches, leading to allergen capture by skin dendritic cells. Mice were orally sensitized to cashew. One week after the end of the sensitization, mice received a patch loaded with cashew protein extract conjugated to Fluoroprobe‐647 (F‐647) for 48 hours. As negative controls, mice received a patch containing excipient. Brachial draining lymph nodes were collected, and cells were isolated and labeled for FACS analysis. The absolute number of cashew‐positive (F‐647‐positive) cells was measured among each DC subsets, (n = 7‐8 per experimental group). Data are median and interquartile ranges of individual values. P values were determined according to the Mann‐Whitney test (**P<.01; ***P<.001)

Figure 4

Kinetic modulation of cashew‐specific antibody response following EPIT to cashew nut in mice. Mice were orally sensitized to cashew. One week after the end of the sensitization, mice were submitted to EPIT. To that end, mice received cashew patches containing 50 µg of cashew protein extract, once a week for up to 16 weeks (in blue). Patches were applied for 48 hours. As negative controls, mice received patches containing excipient (sham, in black) or were kept untreated (naïves, in white). Blood samples were collected before EPIT (B) or every two weeks during treatment to isolate plasma (weeks 2, 4, 6, 8, 10, 12, 14, and 16), as indicated on C panel. Cashew‐specific antibody titers were measured from plasma by indirect ELISA (IgE, panel A) or direct ELISA (IgG1 and IgG2a, panel B and panel C, respectively), (n = 8‐24 per experimental group – 8 mice of each group were challenged at weeks 8 and 12 and killed). Data are median and interquartile ranges of individual values

Figure 5

Evaluation of the level of protection afforded by EPIT against anaphylaxis following 8, 12, or 16 weeks of treatment. Mice were orally sensitized to cashew and treated as described in Figure 4. Following 8 (A, B), 12 (C, D), or 16 (E, F) weeks of EPIT, 8 mice of each group were challenged orally to cashew. (A, C, E) Body temperature was measured every 5 minutes following challenge for 60 minutes. Data are median and interquartile ranges of individual values. (B, D, F) Clinical symptoms were monitored every 5 minutes following challenge for 60 minutes, based on Table S3. (n = 8 per experimental group). Data are mean with SEM of individual values. P values were determined according to the Mann‐Whitney test

Figure 6

Evaluation of mast‐cell activation induced by oral challenge following 8, 12, or 16 weeks of EPIT. Mice were orally sensitized to cashew, treated as described in Figure 4, and finally challenged as described in Figure 5. Blood samples were collected 60 minutes after the challenge to isolate plasma. mMCP‐1 (A, C, E) and mMCP‐7 (B, D, F) concentrations were measured from plasma by ELISA, (n = 8 per experimental group). Data are median with interquartile range of individual values. P values were determined using the Mann‐Whitney unpaired t test (**P<.01; ***P<.001)