Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines

Ana I Manzano, F Javier Cañada, Bárbara Cases, Sofia Sirvent, Irene Soria, Oscar Palomares, Enrique Fernández-Caldas, Miguel Casanovas, Jesús Jiménez-Barbero, José L Subiza, Ana I Manzano, F Javier Cañada, Bárbara Cases, Sofia Sirvent, Irene Soria, Oscar Palomares, Enrique Fernández-Caldas, Miguel Casanovas, Jesús Jiménez-Barbero, José L Subiza

Abstract

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and (1)H-(13)C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.

Keywords: Allergen; Allergoid; Conjugation; Glutaraldehyde; Mannan; Vaccine.

Figures

Fig. 1
Fig. 1
a Representation of the different 1 H NMR spectra for the purified mannan and commercial mannan (Sigma M3640). Both the carbohydrate (5–3 ppm) and the aliphatic protein regions (3-0 ppm) are displayed; b Monosaccharide analysis in the purified mannan (%); c) Amino acids analysis representation in the purified mannan (ng/mg), the black column showing lysine. Results for b and c are expressed as the mean of duplicates ± SD.
Fig. 2
Fig. 2
The percentage of monosaccharides in fraction AM (allergen-treated with glutaraldehyde in the presence of mannan) and fraction POL (allergen-treated with glutaraldehyde in the absence of mannan). POL and AM were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes). Results are expressed as the mean of duplicates ± SD
Fig. 3
Fig. 3
Protein (protein staining: Coomassie blue) and allergen (immunoblotting: IgE) patterns of Phleum pratense allergen preparations as detected in SDS-PAGE and Western blot. Lane N: native allergen extract; Lane POL: glutaraldehyde-treated allergen without mannan; Lane AM: glutaraldehyde-treated allergen with mannan. AM and POL were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes)
Fig. 4
Fig. 4
NMR analysis for the different Phleum pratense allergen preparations. a Superimposition of the different 1H NMR spectra recorded. Both the carbohydrate (5–3 ppm) and the aliphatic protein regions (3-0 ppm) are displayed; b Superimposition of the different DOSY experiments recorded. Carbohydrate (5–3 ppm) region are displayed. AM (allergen-treated with glutaraldehyde in the presence of mannan); POL (allergen-treated with glutaraldehyde in the absence of mannan). AM and POL were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes).
Fig. 5
Fig. 5
NMR analysis for the different Phleum pratense allergen preparations (N, POL, AM) and purified mannan (M). a Superimposition of the different 1H NMR spectra recorded for the different samples. Both the carbohydrate (5–3 ppm) and the aliphatic protein regions (3-0 ppm) are displayed; b Superimposition of the different 1H NMR spectra and DOSY experiments recorded for the different samples. Both the carbohydrate (5–3 ppm) and the aliphatic protein region (3-0 ppm) are displayed. Monodimensional DOSY projections represented in the “Y” axis. AM (allergen-treated with glutaraldehyde in the presence of mannan); POL (allergen-treated with glutaraldehyde in the absence of mannan); N (native allergen extract). AM and POL were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes).
Fig. 6
Fig. 6
NMR analysis for the different Phleum pratense allergen preparations: allergen-treated with glutaraldehyde in the presence of mannan (AM); allergen-treated with glutaraldehyde in the absence of mannan (POL) and purified mannan (M). Superimposition of the different 1H-13C HSQC spectra: AM (blue); POL (black) and M (yellow). Signals from Arabino-galactan following assignment by Brecker et al. [25]; Signals from Mannans following assignment by Vinogradov et al. [24]. Assignment of the carbohydrate anomeric zone: α/βAra (α/β arabinose), βGal (βGalactose), bcM (branched chain mannose), ucM (unsubstituted chain mannose), ibM (2-substituted internal branched mannose), tbM (terminal branched mannose)
Fig. 7
Fig. 7
Stability study as detected by NMR analysis for the different Phleum pratense allergen preparations stored at 4 °C (N, POL, AM). 1H NMR spectra was recorded immediately after their preparations (t0) and four months later (t1). The intensity differences of the key NMR signals between t0 and t1 are highlighted as a percentage (upper part). AM (allergen-treated with glutaraldehyde in the presence of mannan); POL (allergen-treated with glutaraldehyde in the absence of mannan); N (native allergen extract). AM and POL were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes)
Fig. 8
Fig. 8
IgE reactivity with the different Phleum pratense allergen preparations (N, POL, AM) as measured by dot blot. a Quantification of the IgE binding from sera of grass pollen allergic patients by scanning (mean values ± SD); b Dot blot results obtained for 5 individual sera and a pooled serum from patients allergic to grass pollen. N (native allergen extract); POL (allergen-treated with glutaraldehyde in the absence of mannan); AM (allergen-treated with glutaraldehyde in the presence of mannan). AM and POL were fractions recovered in the retentate after tangential ultrafiltration (100 kDa pore size membranes).

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