Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity.
On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.
Vrtala
Susanne
S
Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria.
Focke
Margarete
M
Kopec
Jolanta
J
Verdino
Petra
P
Hartl
Arnulf
A
Sperr
Wolfgang R
WR
Fedorov
Alexander A
AA
Ball
Tanja
T
Almo
Steve
S
Valent
Peter
P
Thalhamer
Josef
J
Keller
Walter
W
Valenta
Rudolf
R
D000485:Allergens / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D000818:Animals; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology*; D005260:Female; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000008:administration & dosage / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D031784:Phleum / Q000235:genetics / Q000276:immunology; D010940:Plant Proteins / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics* / Q000276:immunology; D011058:Pollen / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics* / Q000276:immunology; D014612:Vaccines / Q000008:administration & dosage / Q000138:chemical synthesis / Q000235:genetics* / Q000276:immunology
D000485:Allergens; D007106:Immune Sera; D007074:Immunoglobulin G; C095238:PHLPVI protein, Phleum pratense; D010446:Peptide Fragments; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D007073:Immunoglobulin E