Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2.

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ∼100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4(+) T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

Formulation and Characterization of Orally Dissolving Thin Films containing the German cockroach Blatella germanica (Bla g 2) Allergen.

Allergy and asthma are among the most common chronic diseases of childhood. Cockroach allergy is an important contributor to asthma morbidity, with a prevalence of 17 to 41%. Immunotherapy has been shown to be an effective treatment for other allergies that contribute to asthma, but several factors have limited its use for cockroach allergy. In this work, a sublingual immunotherapy (SLIT) formulation of orally dissolving thin film has been developed for the treatment of hypersensitivity to the German cockroach Bla g 2 allergen. The formulation allows for the incorporation of up to 25 µg/film of the allergen protein, and the film's mucoadhesiveness prolongs the effect of the allergen with the potential for enhanced efficacy. The potency and dose uniformity of the SLIT formulation were characterized by enzyme-linked immunosorbent assay (ELISA), and other physicochemical properties were evaluated by spectroscopic or mechanistic methods. The films were uniform in weight and thickness, and demonstrated substantial physical strength to allow easy manipulation during manufacturing and dosing. The dosage uniformity, in vitro disintegration and in vitro dissolution profiles of the films were within the acceptance criteria in the United States Pharmacopeia. The developed SLIT methodology possesses the potential to significantly improve immunotherapy for both food and inhalant allergies in adults and children.

Human ß-defensin HBD3 binds to immobilized Bla g2 from the German cockroach (Blattella germanica).

Human ß-defensin 3 (HBD3) is a small, well-characterized peptide in mucosal secretions with broad antimicrobial activities and diverse innate immune functions. Among these functions is the ability of HBD3 to bind to antigens. In this study, we hypothesize that HBD3 binds to the allergen Bla g2 from the German cockroach (Blattella germanica). The ability of HBD1 (used as a control ß-defensin) and HBD3 to bind to Bla g2 and human serum albumin (HSA, used as a control ligand) was assessed using the SensíQ Pioneer surface plasmon resonance (SPR) spectroscopy biosensor system. HBD1 was observed to bind weakly to Bla g2, while HBD3 demonstrated a stronger affinity for the allergen. HBD3 was assessed under two buffer conditions using 0.15 M and 0.3 M NaCl to control the electrostatic attraction of the peptide to the chip surface. The apparent K(D) of HBD3 binding Bla g2 was 5.9±2.1 µM and for binding HSA was 4.2±0.7 µM, respectively. Thus, HBD3, found in mucosal secretions has the ability to bind to allergens like Bla g2 possibly by electrostatic interaction, and may alter the ability of Bla g2 to induce localized allergic and/or inflammatory mucosal responses.

Reactivity of German cockroach allergen, Bla g 2, peptide fragments to IgE antibodies in patients' sera.

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.