ABSTRACT
BACKGROUND: Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity. METHODS: A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide-specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross-reactivity between the allergens was investigated by IgE inhibition studies. RESULTS: Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N- and C-terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross-reactivity could be detected. CONCLUSION: Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope-containing area located on similar portions of their structure, but they lack relevant IgE cross-reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen-specific immunotherapy of HDM allergy.
Subject(s)
Allergens/immunology , Antigens, Dermatophagoides/chemistry , Antigens, Dermatophagoides/immunology , Arthropod Proteins/chemistry , Arthropod Proteins/immunology , Cross Reactions/immunology , Epitopes/chemistry , Immunoglobulin E/immunology , Pyroglyphidae/immunology , Animals , Arthropod Proteins/chemical synthesis , Drug Discovery , Epitope Mapping , Epitopes/immunology , Humans , Immunization , Immunoglobulin E/blood , Immunoglobulin G/immunology , Models, Molecular , Molecular Conformation , Protein Conformation, alpha-Helical , Protein Folding , Rabbits , Vaccines, SyntheticABSTRACT
BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.
Subject(s)
Allergens/immunology , Cross Reactions/immunology , Epitopes/immunology , Immunoglobulin G/immunology , Immunotherapy , Pollen/immunology , Allergens/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Antigens, Plant , Calcium/metabolism , Epitopes/chemistry , Humans , Immunoglobulin E/immunology , Models, Molecular , Peptides/immunology , Protein Binding/immunology , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/immunologyABSTRACT
BACKGROUND AND OBJECTIVE: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction. METHODS: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. RESULTS: Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen. CONCLUSIONS: We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.
