Asunto(s)
Alérgenos/administración & dosificación , Esofagitis Eosinofílica/terapia , Eosinófilos/efectos de los fármacos , Inmunoterapia/métodos , Adolescente , Adulto , Alérgenos/química , Alérgenos/inmunología , Alternaria/química , Alternaria/inmunología , Animales , Niño , Alérgenos Animales/química , Alérgenos Animales/inmunología , Esofagitis Eosinofílica/inducido químicamente , Esofagitis Eosinofílica/inmunología , Esofagitis Eosinofílica/patología , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Humanos , Inmunoglobulina E/biosíntesis , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Polen/química , Polen/inmunología , Pyroglyphidae/química , Pyroglyphidae/inmunología , Estudios RetrospectivosRESUMEN
BACKGROUND: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. OBJECTIVE: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. METHODS: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. RESULTS: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. CONCLUSION: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Lipocalinas/inmunología , Multimerización de Proteína , Alérgenos/uso terapéutico , Animales , Anticuerpos/inmunología , Alérgenos Animales/química , Alérgenos Animales/inmunología , Femenino , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/terapia , Inmunoglobulina E/inmunología , Inmunoterapia , Lipocalinas/uso terapéutico , Ratones , Ratones Endogámicos BALB CRESUMEN
Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four ß-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes.