RESUMO
BACKGROUND: Trees of the family Oleaceae (olive and ash) are important allergen sources in Mediterranean countries, Northern and Central Europe, and North America. The major olive pollen allergen Ole e 1 represents the majority of allergenic epitopes in olive pollen and cross-reacts with Fra e 1, the major ash pollen allergen. OBJECTIVE: We sought to develop a safe vaccine for the treatment of Oleaceae pollen allergy. METHODS: We synthesized 5 peptides ranging from 32 to 36 amino acids, which covered the whole sequence of Ole e 1. The IgE and T-cell reactivity of the peptides was compared with that of Ole e 1 by means of dot blot experiments, as well as ELISA, and in proliferation assays. Rabbits were immunized with non-IgE-reactive, keyhole limpet hemocyanin-coupled peptides or Ole e 1. The reactivity of the IgG antibodies with Ole e 1 and their ability to inhibit IgE binding to nOle e 1 was evaluated by means of ELISA. RESULTS: Only the C-terminal Ole e 1 peptide showed IgE binding, whereas the other peptides were nonallergenic. Immunization of rabbits with Ole e 1-derived peptides bound to the carrier molecule keyhole limpet hemocyanin induced in rabbits the production of Ole e 1-specific IgG antibodies, which cross-reacted with Fra e 1, and inhibited olive and ash pollen-sensitized patients' IgE binding to Ole e 1. CONCLUSION: Two non-IgE-binding peptides with low T-cell reactivity from the N-terminus of Ole e 1 were identified that might represent safe vaccine candidates for immunotherapy of Oleaceae pollen allergy.
Assuntos
Antígenos de Plantas/imunologia , Olea/efeitos adversos , Rinite Alérgica Sazonal/prevenção & controle , Vacinação , Adjuvantes Imunológicos/farmacologia , Alérgenos/imunologia , Alérgenos/farmacologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos de Plantas/química , Antígenos de Plantas/farmacologia , Ensaio de Imunoadsorção Enzimática , Hemocianinas/farmacologia , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Olea/imunologia , Peptídeos/síntese química , Peptídeos/imunologia , Peptídeos/farmacologia , Coelhos , Rinite Alérgica Sazonal/imunologiaRESUMO
BACKGROUND: Allergen-specific immunotherapy is clinically effective for the treatment of cat allergy but shows a high rate of side effects. OBJECTIVE: We sought to engineer recombinant fusion proteins for cat immunotherapy that allow reducing both IgE-mediated and T cell-mediated side effects. METHODS: Fusion proteins consisting of the hepatitis B virus-derived PreS domain and 2 nonallergenic Fel d 1-derived peptides were expressed in Escherichia coli and purified. IgE reactivity and allergenic activity of Fel d 1 and the fusion proteins were compared by using IgE-binding assays and basophil activation tests in patients with cat allergy. Mice and rabbits were immunized subcutaneously with Fel d 1 and the fusion proteins to investigate the allergenicity of the vaccines and the development of Fel d 1-specific IgG antibodies. RESULTS: The recombinant fusion proteins showed no relevant IgE reactivity and exhibited more than 1000-fold reduced allergenic activity in basophil activation tests. On immunization of mice and rabbits, the fusion proteins induced Fel d 1-specific IgG antibodies that inhibited the binding of allergic patients' IgE to the allergen without allergic sensitization to Fel d 1. CONCLUSION: The described recombinant fusion proteins exhibit strongly reduced IgE-mediated allergenic activity, contain less than 40% of the Fel d 1 sequence, and thus lack many of the specific T-cell epitopes. Therefore they should represent safe vaccines for the treatment of cat allergy.
Assuntos
Alérgenos/imunologia , Dessensibilização Imunológica/métodos , Glicoproteínas/imunologia , Hipersensibilidade/terapia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Basófilos/imunologia , Estudos de Casos e Controles , Gatos , Glicoproteínas/química , Glicoproteínas/genética , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/biossíntese , Camundongos , Dados de Sequência Molecular , Animais de Estimação/imunologia , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Engenharia de Proteínas , Pirofosfatases/metabolismo , Coelhos , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. OBJECTIVE: We performed molecular, structural, and immunologic characterization of alpha-La. METHODS: Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. RESULTS: According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. CONCLUSIONS: ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.
