Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice

Abstract

Background: Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.

Methodology: BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization.

Results: Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer.

Conclusion: Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.

Figure 1. Experimental design.

Mice were intranasally pretreated with either the chimer (chimer-treat) or sham-treated (poly-sens) 3 times in 7 days intervals followed by 3 intraperitoneal sensitizations with a mixture of rBet v 1, rApi g 1 and rDau c 1 in 2 weeks intervals. Thereafter, mice were sublingually challenged with a mixture of BP, celery and carrot extracts on 3 consecutive days.

Figure 2. IgE-dependent allergen-specific basophil degranulation by sera.

(A) ß-hexosaminidase release from (I.) rBet v 1-, (II.) Api g 1- or (III.) Dau c 1-sensitized mice; each group was i.n. pretreated with rBet v 1 (black bars), rApi g 1 (light-grey bars), rDau c 1 (dark-grey bars) or sham-treated (white bars). (B) ß-hexosaminidase release from mice i.n. pretreated with a mixture of rBet v 1/rApi g 1/rDau c 1 (black bars) compared with poly-sensitized controls (white bars). **p<0.01.

Figure 3. Construction and characterization of the pollen-food-chimer.

(A) Design of the pHis-parallel 2-chimer composed of Bet v 1 protein, flanked by peptides from Api g 1 and Dau c 1. (B) Immunoblot: IgE binding to the chimer of sera from chimer-pretreated/poly-sensitized mice (lane 1) and of BP allergic patients with BPRFA (lane 5, 6, 7), and of Bet v 1 monoclonal antibody (lane 3). Negative controls sera from untreated mouse and non-allergic patient, or buffer control were run in parallel (lane 2, 4, 8. 9).

Figure 4. Antigen-specific cellular and humoral responses in mice pretreated with the chimer and poly-sensitized control mice.

(A) IgE-mediated basophil degranulation. (B) Allergen-specific IgG2a antibodies in sera. (C) Total and antigen-specific IgA antibodies in sera. Chimer-pretreated mice (black bars); poly-sensitized control mice (white bars). *p<0.05, **p<0.01.

Figure 5. Antigen-specific cytokine production in mice pretreated with the chimer and poly-sensitized mice.

(A) IL-5 levels and (B) IFN-γ levels in supernatants of spleen and cervical lymph nodes (CLN) cell cultures after antigen stimulation. Chimer-pretreated mice (black bars); poly-sensitized control mice (white bars). *p<0.05, **p<0.01.

Figure 6. mRNA expression levels of regulatory markers on inductive and local effector sites.

(A) TGF-β, IL-10 and Foxp3 mRNA expression in NALT, and (B) Foxp3 mRNA expression in SLT and BM of chimer-pretreated mice (black bars), shown as relative values in comparison with poly-sensitized controls (white bars). Data are presented as relative ratio of the target genes to the housekeeping gene Alas1.

Figure 7. Effects of blocking antibodies.

(A) Effects of anti-TGF-β, anti-IL-10R and anti-CD25 on levels of IL-5 and IL-4 in supernatants of antigen-stimulated spleen cell cultures. (B) Effects of anti-TGF-β on total and antigen-specific IgA production in sera. Chimer-pretreated mice treated with isotype control antibody (black bars) in comparison with chimer-pretreated and anti-TGF-β treated mice (grey bars), anti-IL10R treated mice (striped bars) and anti-CD25 treated mice (dotted bars). Poly-sensitized control group (white bars). *p<0.05, **p<0.01.

Figure 8. Characterization of Treg cells by Treg suppression assay.

Suppressive activity of Treg cells derived from chimer-treated mice (black bars) and poly-sensitized controls (white bars). Purified CD4⁺CD25⁻ T effector cells (Teff) and CD4⁺CD25⁺ T regulatory cells (Treg) were obtained by cell sorting (MACS), then cultured alone or cocultured in three different ratios (Treg:Teff: 1∶2, 1∶8, 1∶16) in combination with irradiated splenocytes and stimulated by anti-CD3 antibody, before pulsing with [³H] thymidine. The percent suppression mediated by Treg cells is calculated by the following formula: [(cpm of Teff alone – cpm of Teff treated with Treg)/cpm of Teff cells alone]*100.