Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.
The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.
Westritschnig
Kerstin
K
Department of Pathophysiology, Vienna General Hospital, AKH, Medical University of Vienna, Vienna, Austria.
Focke
Margarete
M
Verdino
Petra
P
Goessler
Walter
W
Keller
Walter
W
Twardosz
Anna
A
Mari
Adriano
A
Horak
Friedrich
F
Wiedermann
Ursula
U
Hartl
Arnulf
A
Thalhamer
Josef
J
Sperr
Wolfgang R
WR
Valent
Peter
P
Valenta
Rudolf
R
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D018926:Anti-Allergic Agents / Q000008:administration & dosage / Q000138:chemical synthesis / Q000276:immunology; D052179:Antigens, Plant; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000276:immunology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015550:Cell Degranulation; D045744:Cell Line, Tumor; D003429:Cross Reactions; D003888:Desensitization, Immunologic / Q000379:methods*; D004306:Dose-Response Relationship, Immunologic; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D031784:Phleum / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011058:Pollen / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D051381:Rats; D012882:Skin Tests; D013329:Structure-Activity Relationship; D014612:Vaccines / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics* / Q000276:immunology
D000485:Allergens; D018926:Anti-Allergic Agents; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D007074:Immunoglobulin G; D010446:Peptide Fragments; C468525:Phl p 7 allergen; D010940:Plant Proteins; D014612:Vaccines; D007073:Immunoglobulin E