BACKGROUND:Cupressaceae pollen increasingly causes respiratory allergies worldwide. Carbohydrates are abundant in extracts of these pollens, and the associated allergens are highly glycosylated. However, the contribution of saccharides to the allergenicity of these species remains unknown. METHODS:Juniperus ashei pollen extract was deglycosylated and characterised using SDS-PAGE and immunoblotting. Additionally, N- and O-glycans were purified from the extract, identified, used as inhibitors in IgE-immunoblotting and further analysed via basophil activation tests. The interactions between IgE and J. ashei glycans were analysed using a glycan array. Purified Jun a 1 was treated with β-N-acetylglucosaminidase S and analysed using immunoblotting. The native pollen extract was used to immunise rabbits, and the IgG response was analysed using ELISA and glycan array. RESULTS:Deglycosylation of J. ashei proteins abolished the interaction between IgE and allergens. This effect primarily depends on N-glycans. Purified N-glycans triggered basophil activation in some patients. A biantennary N-glycan with terminal GlcNAc, β-1,2 xylose and core α-1,3 fucose (GnGnXF3) was the most abundant glycan identified. The glycan array confirmed its interaction with IgE. The contribution of terminal N-acetylglucosamines (GlcNAc) to IgE-Jun a 1 interaction was validated. Moreover, effective immunisation of rabbits with the native extract confirmed the immunogenicity of their N-glycans. CONCLUSIONS:The IgE-J. ashei allergen interaction is broadly controlled through N-glycans different from MUXF3. GnGnXF3 exerts an immunogenic effect in humans and rabbits; terminal GlcNAc residues influence its recognition by IgE. These discoveries reinforce the role of N-glycans in the allergic response to J. ashei.

J. ashei pollen is a frequent cause of respiratory allergy. Allergen extracts from this source show high concentration of glycoproteins. We hypothesize a significant allergenic role of glycans bound to its allergens. J. ashei pollen extract was chemically deglycosylated for removing N- and O-glycans. The resulting product was analyzed by SDS-PAGE and IgE-Immunoblotting. Jun a 1 was detected by a polyclonal antibody. N-glycans were excised from J. ashei proteins by PNGase A deglycosylation and purified by ultrafiltration. The remained glycoproteins were then incubated with NaBH4/NaOH for separating O-glycans, and isolated by filtration. IgE-binding capacity of N-glycans, O-glycans and MUXF3 to J. ashei pollen allergens was investigated by Immunoblot inhibition assays. Chemically deglycosylated J. ashei extract showed several bands in a range between 11 and 54 kDa but none of them had IgE-binding capacity. The presence of Jun a 1 was confirmed in the deglycosylated extract. Purified N-glycans produced a dose-dependent inhibition of IgE-binding to J. ashei allergens. O-glycans and MUXF3 did not show IgE-binding inhibition. The results demonstrate that IgE is mainly directed against glucidic component of J. ashei allergens, specifically against N-glycans, and these N-glycans play a crucial role in allergic sensitization to J. ashei.

Quantifying major allergens using immunoassays is essential for evaluating the quality and efficacy of allergenic extracts. However, the direct measurement of allergens with this method cannot be performed in allergoids, due to their immune-physicochemical characteristics. This study set out to develop a method for quantifying Bet v 1 in polymerized birch extracts using mass-spectrometry targeted analysis.

Chemically modified allergens (allergoids) are extensively used for allergen immunotherapy since they have a reduced allergenicity respect to native extracts while maintaining their immunogenicity. However, modification makes characterization of these molecules complicated, requiring specific techniques. The objective was to characterize different depigmented-polymerized pollen extracts. These allergoids have been previously purified (depigmentation process) in order to eliminate allergologically irrelevant low-molecular weight components. Depigmented-polymerized extracts of birch (Betula alba), olive tree (Olea euopaea), grass (Phleum pratense) and pellitory wall (Parietaria judaica) pollens were manufactured from native extracts. The presence of the relevant allergens in the modified molecule was determined by mass spectrometry and the profile of polymerization of the allergoids was determined by high performance-size exclusion chromatography (HPSEC) using a Bio SEC-3 Column (Agilent) in a HPLC system. Peptide sequencing confirmed the presence of the relevant allergens and their isoforms in the allergoids. Thus, allergens of groups 1, 2, 6 and 7 were detected in B. alba, groups 1, 2, 4, 5, 6, 7, 11, 12 and 13 in P. pratense; groups 1, 3, 6, 8, 9 and 10 in O. europaea; and groups 1, 2 and 4 in P. judaica. The HPLC profiles of the allergoids showed high consistency between batches with a decrease in retention time (increase in molecular weight) after polymerization. The depigmented-polymerized allergen extracts of different pollens have been characterized, demonstrating the presence of the relevant allergens and the consistency batch-to-batch in the molecular size of the modified molecule.

RationaleInduction of allergen specific IgG antibodies with capacity to block allergen-IgE interaction has been suggested as mechanism for clinical efficacy, and long term benefit of allergen specific immunotherapy. Induction of these blocking antibodies by depigmented-polymerized allergoids (Dpg-Pol) has been demonstrated previously. The objectives of this study were to investigate the epitope specificity of IgG antibodies induced by Dpg-Pol and native unmodified allergen extracts of birch pollen (Betula alba).MethodsSera from rabbits immunized with native and Dpg-Pol extracts of birch pollen were obtained, and specific IgG to birch, Bet v 1 and Bet t v 2 were measured by ELISA. Linear synthetic peptides of 12 aa, overlapping 6 aa, of Bet v 1 and Bet v 2 were covalently bound to a cellulose membrane, incubated with rabbit sera and with the secondary antibody, and developed by chemiluminiscence.ResultsSerum samples from rabbits immunized with native birch pollen extracts recognised 11 epitopes from Bet v 1, while serum samples from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from animals immunized with native extracts, and 9 epitopes from Dpg-Pol immunized animals. There are some epitopes recognized by both extracts, but some of them are recognized only by the Dpg-Pol extract.ConclusionsDepigmented-polymerized birch pollen allergen extract stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes from the major allergens Bet v 1 and Bet v 2, which may be part of the mechanism of action of this treatment when used for allergen immunotherapy. RationaleInduction of allergen specific IgG antibodies with capacity to block allergen-IgE interaction has been suggested as mechanism for clinical efficacy, and long term benefit of allergen specific immunotherapy. Induction of these blocking antibodies by depigmented-polymerized allergoids (Dpg-Pol) has been demonstrated previously. The objectives of this study were to investigate the epitope specificity of IgG antibodies induced by Dpg-Pol and native unmodified allergen extracts of birch pollen (Betula alba). Induction of allergen specific IgG antibodies with capacity to block allergen-IgE interaction has been suggested as mechanism for clinical efficacy, and long term benefit of allergen specific immunotherapy. Induction of these blocking antibodies by depigmented-polymerized allergoids (Dpg-Pol) has been demonstrated previously. The objectives of this study were to investigate the epitope specificity of IgG antibodies induced by Dpg-Pol and native unmodified allergen extracts of birch pollen (Betula alba). MethodsSera from rabbits immunized with native and Dpg-Pol extracts of birch pollen were obtained, and specific IgG to birch, Bet v 1 and Bet t v 2 were measured by ELISA. Linear synthetic peptides of 12 aa, overlapping 6 aa, of Bet v 1 and Bet v 2 were covalently bound to a cellulose membrane, incubated with rabbit sera and with the secondary antibody, and developed by chemiluminiscence. Sera from rabbits immunized with native and Dpg-Pol extracts of birch pollen were obtained, and specific IgG to birch, Bet v 1 and Bet t v 2 were measured by ELISA. Linear synthetic peptides of 12 aa, overlapping 6 aa, of Bet v 1 and Bet v 2 were covalently bound to a cellulose membrane, incubated with rabbit sera and with the secondary antibody, and developed by chemiluminiscence. ResultsSerum samples from rabbits immunized with native birch pollen extracts recognised 11 epitopes from Bet v 1, while serum samples from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from animals immunized with native extracts, and 9 epitopes from Dpg-Pol immunized animals. There are some epitopes recognized by both extracts, but some of them are recognized only by the Dpg-Pol extract. Serum samples from rabbits immunized with native birch pollen extracts recognised 11 epitopes from Bet v 1, while serum samples from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from animals immunized with native extracts, and 9 epitopes from Dpg-Pol immunized animals. There are some epitopes recognized by both extracts, but some of them are recognized only by the Dpg-Pol extract. ConclusionsDepigmented-polymerized birch pollen allergen extract stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes from the major allergens Bet v 1 and Bet v 2, which may be part of the mechanism of action of this treatment when used for allergen immunotherapy. Depigmented-polymerized birch pollen allergen extract stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes from the major allergens Bet v 1 and Bet v 2, which may be part of the mechanism of action of this treatment when used for allergen immunotherapy.