Abstract

More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope–derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope–derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

Highlights

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  • We investigated in a mouse model to which extent B cell and T cell epitopes derived from the major grass pollen allergen Phl p 1 are involved in boosting secondary IgE responses

  • To investigate the contribution of B and T cell epitopes on secondary allergen-specific IgE Ab responses, we followed the haptencarrier principle established by Benacerraf and constructed a fusion protein consisting of a peptide from the IgE epitope–containing region of the major timothy grass pollen allergen, Phl p 1, and an unrelated carrier protein (i.e., PreS from hepatitis B virus) providing T cell help (Fig. 1) [24]

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Summary

Materials and Methods

A 30-aa peptide derived from the major grass pollen allergen Phl p 1 ranging from aa 212 to 241 (VRYTTEGGTKTEAEDVIPEGWKADTSYESK) was synthesized using a Wang resin from AAPPTec (Louisville, KY) on a Liberty microwave peptide synthesizer from CEM (Darmstadt, Germany). On day 180, spleens were removed under aseptic conditions for preparation of splenocytes and analysis of T cell responses by violet proliferation dye (VPD)450 FACS experiments and thymidine incorporation assays Sera from these mice obtained on day 180 were later used to sensitize rat basophilic leukemia (RBL) cells for testing the molecules used for boosting. To test the ability of the different immunogens (1xP, 2xP, oligo-P, 4xP, PreS, KLH) to cross-link peptide P–specific IgE Abs on basophils, RBL cells were loaded with 1:10 diluted pooled sera obtained on day 180 from mice sensitized and boosted three times with 4xP. To measure peptide-specific release in mice from groups A–F, sera from each mouse group were pooled due to serum limitations and loaded on RBL cells in 1:20 dilutions for 2 h. Results are either shown as box plots with indicated medians using SPSS software version 20.0 or column bar graphs using GraphPad Prism (GraphPad Software) version 5.0

Results
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