Pepsinized cashew proteins are hypoallergenic and immunogenic and provide effective immunotherapy in mice with cashew allergy

Abstract

Background: IgE-mediated allergic reactions to cashews and other nuts can trigger life-threatening anaphylaxis. Proactive therapies to decrease reaction severity do not exist.

Objectives: We aimed to determine the efficacy of pepsin-digested cashew proteins used as immunotherapy in a murine model of cashew allergy.

Methods: Mice were sensitized to cashew and then underwent challenges with digested or native cashew allergens to assess the allergenicity of the protein preparations. Using native or pepsinized cashew proteins, mice underwent oral or intraperitoneal sensitization protocols to determine the immunogenic properties of the protein preparations. Finally, cashew-sensitized mice underwent an immunotherapy protocol with native or pepsinized cashew proteins and subsequent provocation challenges.

Results: Pepsinized cashew proteins elicited weaker allergic reactions than native cashew proteins but importantly retained the ability to stimulate cellular proliferation and cytokine production. Mice sensitized with pepsinized proteins reacted on challenge with native allergens, demonstrating that pepsinized allergens retain immunogenicity in vivo. Immunotherapy with pepsinized cashew allergens significantly decreased allergic symptoms and body temperature decrease relative to placebo after challenge with native and pepsinized proteins. Immunologic changes were comparable after immunotherapy with native or pepsinized allergens: T(H)2-type cytokine secretion from splenocytes was decreased, whereas specific IgG(1) and IgG(2a) levels were increased.

Conclusions: Pepsinized cashew proteins are effective in treating cashew allergy in mice and appear to work through the same mechanisms as native protein immunotherapy.

FIG 1

Allergic reactions to native or pepsin-digested proteins in mice. A, SDS-PAGE gel showing nCSH and pCSH. B, Mice sensitized by means of intraperitoneal injection of nCSH were challenged with nCSH or pCSH at increasing doses and assessed for body temperatures and symptom scores. C, Orally sensitized mice challenged with nCSH or pCSH at increasing doses were assessed for body temperatures and symptom scores. Circles or triangles represent individual mice. Bars represent means with SDs. *P < .05 and **P < .01. Data shown are results after initial experiments to determine optimal challenge doses in groups consisting of 3 mice each.

FIG 2

Cellular responses to nCSH and pCSH. Splenocytes from orally or intraperitoneally sensitized mice (A and B, respectively) were cultured with nCSH or pCSH and assessed for T_H2/T_H1 cytokine production. Bars represent means with SDs by using 4 individual mice per group. Data shown are representative of 2 separate experiments.

FIG 3

In vivo immunogenicity of nCSH and pCSH. Mice were sensitized with nCSH or pCSH by means of oral or intraperitoneal (i.p.) administration. Orally sensitized mice were assessed for specific IgE (A) and challenged with nCSH to determine allergic status by change in body temperature (B). Mice sensitized intraperitoneally were challenged with nCSH and assessed for symptom scores (C) and body temperature changes (D). Circles represent individual mice. Bars represent means with SDs.

FIG 4

Comparison of IgE binding to nCSH and pCSH in mice sensitized by means of intraperitoneal (i.p.) injection or oral gavage. Mice were sensitized with nCSH by means of either intraperitoneal injection or oral gavage, and serum IgE levels were measured against nCSH or pCSH by using ELISA. Absolute levels of IgE are shown, as well as ratios of anti-nCSH/anti-pCSH IgE. Bars represent means with SDs. Circles represent individual murine sera. **P < .01.

FIG 5

Immunotherapy (IT) with nCSH and pCSH in orally sensitized mice. Mice were sensitized with nCSH and then administered nCSH, pCSH, or placebo immunotherapy. Naive mice are nonsensitized control animals. A, Challenges with 0.25 mg of nCSH were assessed for symptom scores and body temperatures. B, Challenges with 1.0 mg of pCSH were assessed for symptom scores and body temperatures. Circles represent individual mice. Bars represent means with SDs. **P < .01 versus placebo, #P < .05 versus pCSH immunotherapy, and ##P < .01 versus pCSH immunotherapy. Data shown are combined results from 2 independent experiments using 5 mice per group.

FIG 6

Humoral and cellular profiles after immunotherapy (IT) in orally sensitized mice. Mice sensitized orally with nCSH were given nCSH, pCSH, or placebo immunotherapy. Serum levels of anti-nCSH and anti-pCSH IgG₁ (A), IgG_2a (B), and IgE (C) after immunotherapy are shown. Cytokines secreted from splenocytes of the immunotherapy groups are also shown (D). Bars represent means with SDs. *P < .05 versus placebo, **P < .01 versus placebo, #P < .05 versus pCSH immunotherapy, and ##P < .01 versus pCSH immunotherapy. Data shown are representative of 2 experiments and include 5 mice per group.