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

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 1H-13C 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.

Oral myeloid cells uptake allergoids coupled to mannan driving Th1/Treg responses upon sublingual delivery in mice

“…PM‐allergoids are the result of coupling polymerized allergoids to nonoxidized mannan from Saccharomyces cerevisae in such a way that the mannan structure is duly preserved 18, 22. Our results indicate that PM‐allergoids induce a faster and stronger immune response than that obtained with the native allergen, both at the cellular and humoral levels.…”

“…It is noteworthy to mention that every response assessed was always against the native allergens, irrespectively of the allergen preparation used for immunization. The immune response obtained with PM‐allergoids is achieved in spite of their high molecular weight,18 early suggested to be a serious impediment for the immunization through the sublingual route 23. Supporting this route also for PM‐allergoids is the response obtained in submandibular LNs, the primary draining lymph nodes of sublingual tissue 24.…”

“…Grass pollen ( Phleum pratense) allergens and mannan ( Saccharomyces cerevisae ) were obtained and purified as described 18. Unmodified native allergens (N) were coupled to nonoxidized mannan (1:0.5 ratio) exactly as described18 to render polymerized allergoid‐mannan complexes (PM).…”

“…Unmodified native allergens (N) were coupled to nonoxidized mannan (1:0.5 ratio) exactly as described18 to render polymerized allergoid‐mannan complexes (PM). For control purposes, PM‐allergoids were further oxidized (PM‐OX) with sodium periodate as described 16…”

“…PM‐allergoids are taken up by DCs very rapidly through a receptor‐mediated mechanism involving C‐type lectin receptors 16. Besides their better uptake, these glycoconjugates are rendered hypoallergenic16, 17, 18 and able to activate DCs to promote the induction of functional FOXP3 + Treg cells,16 thus with improved features for allergen immunotherapy 19. These properties may be especially relevant for SLIT due to the very short time the allergens are available to mucosal DCs, which might well explain the high allergen doses needed for clinical efficacy in SLIT 12, 14.…”