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To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects. We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity. Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2–sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract. Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P <.01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P <.05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice. By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated. [ABSTRACT FROM AUTHOR]

• Fish parvalbumins are internalized by bronchial epithelial cells and transported across the epithelial monolayers. • Parvalbumins do not affect the epithelial barrier properties. • Low molecular weight components from fish meat modify physical and immunological properties of the epithelial barrier. • Low molecular weight components from bony versus cartilaginous fish display different effects on epithelial cells. • Different effects on epithelium may play a role in differential sensitizing capacities of the two fish classes. The prevalence of fish allergy among fish-processing workers is higher than in the general population, possibly due to sensitization via inhalation and higher exposure. However, the response of the bronchial epithelium to fish allergens has never been explored. Parvalbumins (PVs) from bony fish are major sensitizers in fish allergy, while cartilaginous fish and their PVs are considered less allergenic. Increasing evidence demonstrates that components other than proteins from the allergen source, such as low molecular weight components smaller than 3 kDa (LMC) from pollen, may act as adjuvants during allergic sensitization. We investigated the response of bronchial epithelial cells to PVs and to LMC from Atlantic cod, a bony fish, and gummy shark, a cartilaginous fish. Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with fish PVs and/-or the corresponding fish LMC. Barrier integrity, transport of PVs across the monolayers and release of mediators were monitored. Intact PVs from both the bony and the cartilaginous fish were rapidly internalized by the cells and transported to the basolateral side of the monolayers. The PVs did not disrupt the epithelial barrier integrity nor did they modify the release of proinflammatory cytokines. In contrast, LMC from both fish species modified the physical and immunological properties of the epithelial barrier and the responses differed between bony and cartilaginous fish. While the barrier integrity was lowered by cod LMC 24 h after cell stimulation, it was increased by up to 2.3-fold by shark LMC. Furthermore, LMC from both fish species increased basolateral and apical release of IL-6 and IL-8, while CCL2 release was increased by cod but not by shark LMC. In summary, our study demonstrated the rapid transport of PVs across the epithelium which may result in their availability to antigen presenting cells required for allergic sensitization. Moreover, different cell responses to LMC derived from bony versus cartilaginous fish were observed, which may play a role in different allergenic potentials of these two fish classes. [ABSTRACT FROM AUTHOR]

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli , purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100 ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2. [ABSTRACT FROM AUTHOR]