Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines
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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.
KeywordsMannan Allergen Allergoid Glutaraldehyde Conjugation Vaccine
The immunotherapy of IgE-mediated allergic diseases is based on the administration of increasing amounts of allergens to desensitize allergic patients (allergen vaccines). For immunotherapy to be effective, high doses of allergens have to be administered . This raises major safety concerns due to the sensitivity of the patients to the allergens that the vaccine contains .
Immunotherapy with modified allergens to render them hypoallergenic (allergoids) is increasingly being used, since the safety profile of allergoids allows to a faster and simpler dosing than with native (non-modified) allergens [2, 3]. Glutaraldehyde is the most widely used agent for allergoid formation [4, 5]. By means of its two reactive aldehyde groups, it cross-links the allergen proteins through the ε-amino groups of lysine residues. This reaction results in allergen polymerization, with the concomitant loss of accessibility of IgE antibodies to the allergen epitopes, i.e., antibody binding sites, .
The goal of allergy vaccines is to induce a therapeutic immune response against the corresponding allergens. The main initiators of such a response are dendritic cells (DCs), which are continuously sampling antigens from the microenvironment by receptor-mediated endocytosis, micropinocytosis and phagocytosis . C-type lectin receptors (CLRs) are pivotal for recognizing glycans, in a calcium-dependent manner . Key examples are mannose receptor (MR), Dectin-2 and DC-SIGN, which preferentially recognize mannose residues . Therefore, antigen conjugation with mannan, as a source of polymannoses, has been proposed to increase the antigen uptake by DCs , including allergens . While this concept is of paramount interest for vaccine development, the notion is even more evident when considering hypoallergenic polymerized allergens, since it has been claimed that these polymers are not efficiently captured by DCs .
Conventional methodologies for coupling mannan to proteins require, as a first step, its prior oxidation (oxidized mannan) to release reactive aldehyde groups (−CHO) able to bind to the protein amino groups . This approach is however not suitable for conjugating mannan to allergoids, because the dramatic reduction of free amino groups once the protein has reacted with glutaraldehyde .
Here we show a novel approach to obtain allergoid-mannan glycoconjugates. Our concept is based on using just a glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone of mannan. Analytical and structural studies show the feasibility of such an approach, which results in a high molecular weight and stable structure with a reduced IgE binding reactivity suitable to be used for allergen immunotherapy.
Material and methods
Defatted grass pollen grains from Phleum pratense (Iberpolen, Jaén, Spain) were extracted overnight with phosphate buffered saline, pH 7.2 (PBS) and submitted to tangential flow ultrafiltration (cut off pore size, 100 kDa). The enriched allergen fraction obtained in the filtrate was dialyzed with distilled water and lyophilized in small aliquots until used. Total protein content was measured by the Bradford assay using serum albumin as standard (Bio-Rad Laboratories, Madrid, Spain).
Mannan from S. cerevisae
Mannan was obtained as described [14, 15] with slight modifications. Briefly, mannan was extracted from yeast (Saccharomyces cerevisae; Lesaffre Ibérica, Madrid, Spain) in hot citrate buffer (0.02 M; pH 7.0) during 90 min. The extract was precipitated with ethanol and dialyzed against distilled water. Mannan was precipitated in the presence of cetavlon (Sigma-Aldrich, Madrid, Spain) (50 % v/v) after several hours in a shaker by adding 2 % borate sodium (pH 8.8). The precipitate was collected by centrifugation and washed twice with 2 % acetic acid in ethanol plus a final wash with 100 % ethanol. Once re-dissolved and dialyzed in distilled water, it was applied to a DEAE-Sephadex A-50 column equilibrated with 0.02 M Tris-HCl buffer (pH 7.5). A linear gradient from 0 to 0.5 M NaCl was used. Mannan-containing fractions (M) were pooled, dialyzed extensively against distilled water and lyophilized in aliquots until used.
Allergens from grass pollen Phleum pratense were polymerized and conjugated with mannan in a single step as follows. Glutaraldehyde (25 %, Sigma) was added to a solution (final concentration 25 mM) containing a mixture of the allergen and mannan in PBS. This mixture was made at different allergen:mannan ratios (1:4; 1:1; 1:0.5; 1:0.3; 1:0.15), using a fix protein amount and different amounts of freeze-dried mannan. Reaction was performed during 6 h at 4 °C in continuous stirring and stopped with glycine (1.25 M), followed by tangential flow filtration with distilled water (membrane cut off, 100 kDa) to remove free allergen and mannan molecules, virtually all of them below that size. Allergen-mannan (AM) conjugates were recovered in the concentrated retentate (>100 kDa fraction) that was further lyophilized until use. Total carbohydrates and protein content of reconstituted samples were measured by the anthrone  and Bradford  assays, respectively.
For control purposes, one part of the same allergen extract remained untreated (native allergen, N) or subjected to the above protocol but without mannan to obtain a conventional mannan-free polymerized allergen (POL).
Gel electrophoresis (SDS-PAGE) and immunoblotting
Every allergen sample (N, POL and AM) was submitted to protein separation in 12.5 % polyacrylamide gels under denaturalizing conditions with sodium dodecyl sulfate and Coomassie blue staining. Immunoblots were performed transferring the proteins separated by electrophoresis to cellulose nitrate membranes (Bio Rad, Germany). The membranes were blocked with 5 % bovine serum albumin in PBS-0.1 % Tween 20 and incubated with a pooled serum from grass allergic patients. Afterwards, they were incubated with an anti-human IgE monoclonal antibody conjugated with peroxidase (Southern Biotech, USA) at a 1/2000 dilution. ECL chemiluminescence system was used for reaction development (GE- Healthcare, USA).
Amino acid analyses
Samples adjusted at 1 mg/mL in protein were hydrolyzed with HCl 6 N, during 24 h under vacuum at 110 °C. Amino acid content was assessed by a quantitative amino acid analyzer (Biochrom 30; Biochrom Ltd., Cambridge, UK). The process requires the separation of the amino acids by cation exchange chromatography and derivatization with ninhydrin .
Neutral sugars were analyzed by gas chromatography in the form of their alditol acetates . The samples (1 mg) containing polysaccharides were first hydrolyzed with 3 M tri-fluoroacetic acid (121 °C, 1 h). The released monosaccharides were then converted into their corresponding alditol acetates by reduction with NaBH4 (Sigma) and subsequent acetylation. Identification and quantification were performed by gas-liquid chromatography on a 6890A instrument (Agilent Technologies, Santa Clara, California, USA) equipped with a flame-ionization detector, using a HP5 fused silica column with He as the carrier gas. Identification was performed on the basis of the coincidence of the retention time of the sample components with those previously measured for known monosaccharide reference standards analyzed under identical conditions and using inositol as internal standard.
Nuclear magnetic resonance (NMR) studies
NMR spectra were obtained for samples (4 mg/mL in D2O) at 298 K. Standard 1H NMR and 2D-NMR experiments (TOCSY, HSQC, DOSY) were employed using Bruker Avance 500, 600 and 700 MHz spectrometers (Bruker Ltd., Germany). For heteronuclear 2D-NMR (HSQC), experiments were acquired with 2 K points in a spectral width of 9 ppm in the 1H dimension and 110 ppm, center at 55 ppm, in the 13C dimension and 256 increments. A relaxation delay of 1 s, and a J-evolution delay corresponding to a J value of 155 Hz were used.13C decoupling was achieved by the WALTZ scheme. Standard conditions were employed for the homonuclear 2D-NMR (TOCSY) experiments, with a 50 ms mixing time. The two dimension diffusion ordered spectroscopy (2D-DOSY) experiments were carried out by recording 64–128 scans for each gradient step, a linear gradient of 16 steps between 2 % and 95 %, a diffusion time (big delta) between 0.2 and 0.4 s, and the length of the diffusion encoding gradient pulses (little delta) between 2-4 ms. Dextran markers of different MW were used to perform a calibration curve to obtain the diffusion coefficients (m2 s−1) as a function of the MW. . All spectra were processed with the protocols implemented in Topspin software (Bruker Ltd). These samples (4 mg/mL in D2O) were stored in closed vials at 4 °C during four months to assess the stability of allergen-mannan glycoconjugates (AM) in comparison to native (N) or just polymerized (POL) preparations.
Specific IgE immunodetection assay
IgE reactivity to the different allergen samples (N, POL and AM) was tested by an immunodetection assay as previously described . Briefly, a total protein amount of 30 μg of the different allergen preparations were placed onto nitrocellulose membranes using a Whatmann Univac vacuum manifold system (GE Healthcare, Canton MA, USA) for allergen binding. The membranes were neutralized with a blocking solution (PBS-0.1 % Tween 20; 3 % defatted milk protein preparation) and incubated (16 h at 4 °C) with individual sera diluted 1/10 from grass pollen allergic patients or with a pool of sera at the same dilution. Afterwards, the membranes were washed with PBS-0.1 % Tween 20 and incubated 1 h at room temperature with anti-human IgE monoclonal antibody diluted 1/5000 followed by a 1 h incubation with a rabbit anti-mouse monoclonal antibody conjugated with peroxidase diluted 1/2000 (Dako, Barcelona, Spain). After washing the membranes with PBS-0.1 % Tween 20, the ECL chemiluminescence system was used for reaction development (GE-Healthcare, Canton MA, USA). Volummograms of the reactive spots were analyzed by scanning densitometry using FUJI FILM MultiGauge v3.0 software.
Allergen conjugation with mannan from S. cerevisae
Analytical studies of the AM fraction
Structural studies of the AM fraction
Stability of allergen-mannan glycoconjugates
IgE reactivity with allergen-mannan glycoconjugates
Coupling of allergens to mannan has been suggested as a way to enhance the allergen uptake by DCs and therefore the efficacy of immunotherapy with allergens . This approach could be even more interesting in the case of allergoids, since it has been claimed that glutaraldehyde-modified allergens are less immunogenic than native allergens (non-modified) due to a lower uptake by DCs . The purpose of this study was to obtain glutaraldehyde-modified allergens conjugated to mannan, taking into account that the conventional conjugation procedure using oxidized mannan was not feasible due to the lack of free amino groups in the allergoids .
Here we show that stable allergoids conjugated with mannan can be produced by treating with glutaraldehyde a mixture of allergen and non-oxidized mannan. These glycoconjugates are likely formed through the reaction of the di-aldehyde with the lysine free amino groups of proteins derived from both the allergen and the mannoprotein, i.e., the protein tail linked to the mannan carbohydrate backbone ; thus, bridging both molecules by means of glutaryl-diimine bonds. The presence of detectable lysine in our purified mannan preparation (almost 1 μg per mg of mannan) supports such a mechanism. Of note, lysine was also present in all commercially available mannan from Saccharomyces cerevisae tested so far (data not shown). The structural studies by NMR show a clear interaction between the allergen and mannan after glutaraldehyde treatment, resulting in high molecular weight complexes. In fact, the broadening of the signals in the allergen-mannan samples indicates a size increase of the complexes as also noted by SDS-PAGE. This is corroborated in translational diffusion DOSY NMR spectra. These complexes showed the lowest diffusion coefficient, as compared with untreated native allergen or glutaraldehyde-treated allergen without mannan, indicating the presence of a molecular entity of larger size. This also evidenced a stable interaction between the allergoid and mannan, since both parts of the spectrum (the carbohydrate and protein areas) show the same diffusion coefficient. Additionally, NMR studies based on HSQC (heteronuclear correlation 13C-1H) experiments confirmed such interaction, since the allergen-mannan conjugates had the characteristic carbohydrate signals of mannan and also of the intrinsic carbohydrates of the allergen. It should be noted that, in HSQC experiments, every C-H pair of the molecular entity displays a signal with well-defined position, depending on its chemical environment. Thus, the complete set of signals compose a molecular fingerprint, which can be ascribed to a given component . Interestingly, the allergen-mannan glycoconjugates prepared with glutaraldehyde seem to be fairly stable during at least several months, as deduced by their subtractive spectrum by NMR. In fact, they were more stable than the untreated native allergen or the glutaraldehyde-treated allergen without mannan. The remarkable structural stability of allergen-mannan glycoconjugates is clearly an advantage from the perspective of a vaccine development, because the possibility of a longer shelf life.
We have also shown that the allergen-mannan glycoconjugates prepared with glutaraldehyde display a reduced allergenicity. The extent of reactivity of these glycoconjugates with specific IgE was even lower than that observed for the conventional allergoids, i.e., polymerized allergens without mannan. The higher molecular weight of allergen-mannan conjugates may explain this fact, since it is considered that the reduced IgE reactivity with the allergoids depends on the loss of accessibility (steric hindrance) of the antibodies to the allergen epitopes after polymerization .
It is important to note that the conjugation approach we describe here preserves the carbohydrate structure of mannan without denaturation, in contrast to the widely used oxidative methods . Oxidation breaks the mannopyranose rings within the polymannose backbone producing remarkable structural consequences [30, 31, 32], that may affect its biological properties such as the interaction with antibodies and lectins [33, 34, 35]. This is of utmost importance when considering the interaction of mannoses with their corresponding lectin receptors on DCs. In fact it has been already shown that the state of oxidation of mannan may modify the behavior of these cells upon activation . Functional studies from ourselves point to the same direction (Sirvent et al., submitted for publication) supporting the suitability of the conjugation approach we describe here to produce neoglycoconjugates with mannan to be used in the development of novel vaccines.
This work was supported by grant IDI-20110410 from the Centre for the Development of Industrial Technology (CDTI- MINECO, Spain).
- 1.Casale, T.B., Stokes, J.R.: Immunotherapy: what lies beyond. J. Allergy Clin. Immunol. 133(3), 612–619: quiz 620 (2014). doi: 10.1016/j.jaci.2014.01.007
- 5.Subiza J., Feliu A., Subiza J.L., Uhlig J., Fernandez-Caldas E.: Cluster immunotherapy with a glutaraldehyde-modified mixture of grasses results in an improvement in specific nasal provocation tests in less than 2.5 months of treatment. Clin. Exp. Allergy. 38(6), 987–994 (2008). doi: 10.1111/j.1365-2222.2008.02995.x CrossRefPubMedGoogle Scholar
- 6.Sallusto F., Cella M., Danieli C., Lanzavecchia A.: Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182(2), 389–400 (1995)CrossRefPubMedGoogle Scholar
- 10.Weinberger E.E., Himly M., Myschik J., Hauser M., Altmann F., Isakovic A., Scheiblhofer S., Thalhamer J., Weiss R.: Generation of hypoallergenic neoglycoconjugates for dendritic cell targeted vaccination: a novel tool for specific immunotherapy. J. Control. Release. 165(2), 101–109 (2013). doi: 10.1016/j.jconrel.2012.11.002 PubMedCentralCrossRefPubMedGoogle Scholar
- 11.Heydenreich B., Bellinghausen I., Lorenz S., Henmar H., Strand D., Wurtzen P.A., Saloga J.: Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells. Immunology. 136(2), 208–217 (2012). doi: 10.1111/j.1365-2567.2012.03571.x PubMedCentralCrossRefPubMedGoogle Scholar
- 13.Silva C.J.S.M., Fernanda Sousa F., Georg Gübitz G., Cavaco-Paulo A.: Chemical modifications on proteins using glutaraldehyde. Food Technol. Biotechnol. 42(1), 51–56 (2004)Google Scholar
- 22.Haavik S., Smestad Paulsen B., Wold J.K.: Glycoprotein allergens in pollen of timothy. I. Investigation of carbohydrates extracted from pollen of timothy (Phleum pratense) and purification of a carbohydrate-containing allergen by affinity chromatography on concanavalin A-sepharose. Int. Arch. Allergy Appl. Immunol. 78(2), 197–205 (1985)CrossRefPubMedGoogle Scholar
- 25.Brecker L., Wicklein D., Moll H., Fuchs E.C., Becker W.M., Petersen A.: Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.). Carbohydr. Res. 340(4), 657–663 (2005). doi: 10.1016/j.carres.2005.01.006 CrossRefPubMedGoogle Scholar
- 27.Claridge T.D.W.: High-Resolution NMR techniques in organic chemistry. Tetrahedon Organic Chemistry., vol. 27, Second edn. Elsevier, Amsterdam, The Netherlands (2009)Google Scholar
- 29.Wilson M.B., Nakane P.P.: Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: Knapp W., Holubar K., Wick G. (eds.) Immunofluorescence and related staining techniques, pp. 215–224. Elsevier, North Holland Biomedical, Amsterdam (1978)Google Scholar
- 31.Durana R., Lacik I., Paulovicova E., Bystrickya S.: Functionalization of mannans from pathogenic yeasts by different means of oxidations - preparation of precursors for conjugation reactions with respect to preservation of immunological properties. Carbohydr. Polym. 63, 72–81 (2006)CrossRefGoogle Scholar
- 36.Sheng K.C., Pouniotis D.S., Wright M.D., Tang C.K., Lazoura E., Pietersz G.A., Apostolopoulos V.: Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells. Immunology. 118(3), 372–383 (2006). doi: 10.1111/j.1365-2567.2006.02384.x PubMedCentralCrossRefPubMedGoogle Scholar
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