Identification of T-Cell Epitopes of Phl p 5a as a Basic Requirement for Construction of Hypoallergenic Mutants

Abstract

RATIONALE: Recombinant Phl p 5a derivatives are promising candidates for an allergen cocktail for specific immunotherapy based on hypoallergenic Timothy grass major allergens. An important prerequisite for construction of such mutants is identification of T-cell epitopes on Phl p 5a.METHODS: T-cell lines were raised from PBMC of grass pollen allergic subjects with natural or recombinant Phl p 5a. T-cell epitopes were analyzed with synthetic dodeca peptides spanning the whole sequence of 288 amino acids of rPhl p 5a overlapping by 3 residues. T-cell reactivity was measured by proliferation assays using 3H-Thymidine.RESULTS: Results from 30 TCL of 16 different grass pollen allergics are included. Two dominant regions of T-cell reactivity are found in the N-terminal part of Phl p 5a at positions 13-27 and 40-54. Four further dominant regions are located in the C-terminal half of the molecule at positions 160-174, 172-186, 196-228 and 229-252. Further peptides with lower frequencies of T-cell reactivity were found between positions 55-162. The T-cell reactivity of a mutant with deletions at positions 94-113 and 175-190 coincides with these findings.CONCLUSIONS: Five regions of frequent T-cell reactivity could be determined on Phl p 5a, together with one region with T-cell reactivity of lower frequency and 5 - 6 regions with no T-cell reactivity. The latter segments of the molecule are those where genetic engineering might produce promising mutants with a changed conformation and reduced IgE-reactivity. RATIONALE: Recombinant Phl p 5a derivatives are promising candidates for an allergen cocktail for specific immunotherapy based on hypoallergenic Timothy grass major allergens. An important prerequisite for construction of such mutants is identification of T-cell epitopes on Phl p 5a. METHODS: T-cell lines were raised from PBMC of grass pollen allergic subjects with natural or recombinant Phl p 5a. T-cell epitopes were analyzed with synthetic dodeca peptides spanning the whole sequence of 288 amino acids of rPhl p 5a overlapping by 3 residues. T-cell reactivity was measured by proliferation assays using 3H-Thymidine. RESULTS: Results from 30 TCL of 16 different grass pollen allergics are included. Two dominant regions of T-cell reactivity are found in the N-terminal part of Phl p 5a at positions 13-27 and 40-54. Four further dominant regions are located in the C-terminal half of the molecule at positions 160-174, 172-186, 196-228 and 229-252. Further peptides with lower frequencies of T-cell reactivity were found between positions 55-162. The T-cell reactivity of a mutant with deletions at positions 94-113 and 175-190 coincides with these findings. CONCLUSIONS: Five regions of frequent T-cell reactivity could be determined on Phl p 5a, together with one region with T-cell reactivity of lower frequency and 5 - 6 regions with no T-cell reactivity. The latter segments of the molecule are those where genetic engineering might produce promising mutants with a changed conformation and reduced IgE-reactivity.