Dragana Stanic-Vucinica,
Marija Stojadinovicab,
Ivana Mirkovc,
Danijela Apostolovicd,
Lidija Burazere,
Marina Atanaskovic-Markovicfg,
Milena Kataranovskich and
Tanja Cirkovic Velickovic*aij
aUniversity of Belgrade, Faculty of Chemistry, Center of Excellence for Molecular Food Sciences, Department of Biochemistry, Studentski trg 16, 11000 Belgrade, Serbia. E-mail: tcirkov@chem.bg.ac.rs; Fax: +381 11 333 6608; Tel: +381 11 333 6608
bUniversity of Belgrade, Faculty of Chemistry, Department of Biochemistry, Studentski trg 16, Belgrade, Serbia
cInstitute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar despota Stefana 142, Belgrade, Serbia
dInnovation Center, Faculty of Chemistry Ltd, Studentski trg 16, Belgrade, Serbia
eInstitute of Virology, Vaccines and Sera – Torlak, Vojvode Stepe 458, Belgrade, Serbia
fUniversity Children's Hospital “Tirsova”, Department of Allergology and Pulmology, University of Belgrade, Tirsova 10, Belgrade, Serbia
gUniversity of Belgrade, School of Medicine, Dr Subotica 8, Belgrade, Serbia
hUniversity of Belgrade, Faculty of Biology, Studentski trg 16, Belgrade, Serbia
iGhent University Global Campus, Incheon, Korea
jGhent University, Faculty of Bioscience Engineering, Ghent, Belgium
First published on 2nd September 2016
Modified allergens are a safer and more efficient alternative to natural allergens for specific immunotherapy. As the modification of an allergen can diminish its immunogenicity due to the alteration of T-cell epitopes, in this paper we study the effects of a reversible chemical modification of Art v 1, the main allergen of mugwort pollen, on its allergenicity and immunogenicity. Modification of Art v 1 by cis-aconitylation into a polyanionic derivative (CAA) did not result in any significant structural alteration. However, IgE-binding epitopes on CAA were blocked, resulting in a reduced IgE-binding and basophil activation. Both proteins induced proliferation of CD3+CD4+ T-cells in mugwort-allergic patients, but only unmodified allergens increased IL-4, IL-5 and IL-10 production. Rabbit and mouse anti-CAA antibodies exhibited cross-reactivity with native allergens and blocked human IgE-binding to Art v 1. Degradation of CAA by lysosomal fraction enzymes resulted in a similar set of peptides, harboring MHC class II-binding peptides, as unmodified proteins. Thus, cis-aconitylation modified Art v 1 had a significantly reduced allergenicity, whereas its immunogenicity was completely preserved. Acid-environment-responsive modification, which releases a full repertoire of native allergen epitopes within a particular site, can be considered a smart drug delivery system, which is able to deliver a therapeutically-effective dose in a controlled manner, and minimizes adverse side effects.
Current ASIT protocols are long and have a risk of anaphylactic reactions when native allergens are used. Therefore, current research is aimed at decreasing the risk of side effects while maintaining or improving the efficacy of ASIT.2
Different strategies for increasing the safety and efficacy of immunotherapies are under development and these strategies target B-cells, T-cells or antigen-presenting cells (APCs). Hypo-allergenic recombinant variants of major allergens aim at reducing side-effects of IgE-mediated reactions, while inducing T-cell tolerance and generation of IgG4 blocking antibodies.2,3 T-Cell peptides of allergens that are devoid of conformational B-cell epitopes, but long enough to bind to T-cell receptors, are examined for induction of tolerance.4 Small peptides representing minor B-cell epitopes fused to an immunogenic carrier protein aim at inducing blocking antibodies against conformational epitopes, but without the activation of allergen-specific T-cells.3 In addition, there are strategies that aim at dendritic cells (DCs) and endolysosomal compartments (ELC) of APCs in particular, i.e. the so-called LAMP-vax™ DNA vaccines, which produce the encoded allergen sequence inside the cell as part of a fusion protein, with a lysosomal-associated membrane protein (LAMP)3 and neoglycoallergens are delivered to dendritic cells via specific receptors.5
Among different approaches for AIT improvement, the use of chemically modified hypoallergenic derivatives stands out as a clinically effective strategy that has been proven in double-blind placebo-controlled studies for decades.6 Ideally, a modified allergen suitable for use in AIT should have its IgE binding epitopes destroyed by modification in order to diminish (or reduce) IgE-binding and IgE-mediated side effects, while structural motifs necessary for T-cell recognition should be preserved. Since many of the B- and T-cell epitopes in allergens are virtually identical, or shared, chemical modification in an uncontrollable manner inevitably destroys certain T-cell epitopes and may lead to a reduced or even diminished capacity for a modified protein to induce allergen-specific immune responses.4–7
In this study we tested a hypothesis that an acid-sensitive polyanionic derivative of a pollen allergen will generate a full repertoire of native B- and T-cell epitopes during acidification in ELC and efficiently raise cross-reactive antibodies to the native protein (blocking antibodies), while also bypassing IgE-mediated reactions of effector cells due to masking of the IgE-epitopes. Our approach included modification of the major mugwort pollen allergen, Art v 1,7 by an acid-sensitive modification: cis-aconitylation of lysine ε-amino groups in the protein.8,9
We demonstrated that our approach created a hypoallergenic protein with a fully preserved fold and intact repertoire of T-cell epitopes. Profiling endolysosomal degradation of proteins in APCs by mass spectrometry identified Art v 1-derived peptides and proved the controlled delivery of a full repertoire of T-cell epitopes in ELC.
The obtained cis-aconitylated Art v 1 (CAA) had approximately 80% modified amino groups, dramatically reduced pI and low stability in acidic conditions.9
Fluorescence measurements were performed using a Horiba Scientific Fluoromax-4 Spectrofluorometer (Horiba, Kyoto, Japan). For intrinsic tryptophan fluorescence measurements Art v 1 samples were diluted in 10 mM potassium phosphate buffer (pH 6.5) to give a final concentration of 0.5 μM and emission spectra were recorded with an excitation wavelength of 280 nm (5 nm slit width). For hydrophobic ligand binding experiments, the fluorescence spectra of the Art v 1 solutions (16 μM in 10 mM potassium phosphate buffer pH 6.5) saturated using 80 μM of 8-anilino-1-naphthalenesulfonic acid (ANS, Sigma-Aldrich) in 10 mM sodium phosphate buffer pH 8 were recorded with an excitation wavelength of 350 nm.
The antibodies against Art v 1, CAA and irrelevant proteins (kiwi fruit protein extract) were raised in rabbits according to Harboe & Ingild11 and as previously described for acetylated Art v 1.12 All experiments were approved by the Ethics Committee for Animal Experimentation at Belgrade University. Two rabbits were immunized subcutaneously with each of the two proteins and the serum obtained by pooling serum specimens of two rabbits was used for experiments. Before the first immunization, serum samples were taken as the reference point, and then every 2 weeks after 51 days from the start of immunization. For direct ELISA, Art v 1 and CAA were coupled to the microtiter plates (Nunc, MaxiSorp, Roskilde, Denmark, 5 μg mL−1) and the total IgG levels were determined by ELISA using anti-rabbit IgG (whole molecule) alkaline phosphatase (AP) labeled antibodies (Sigma-Aldrich) and 4-nitrophenyl phosphate (4-NPP, Sigma-Aldrich) as the substrate. Rabbit serum was diluted 1:
100 in a diluting buffer consisting of 0.5% BSA in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20 (TTBS), and then double fold serial dilutions were performed in the same buffer. In total, 12 double fold serial dilutions were done. A control series was done on the same plates with the rabbit antiserum raised against kiwi fruit extract proteins. Absorbance was measured at 405 nm after adjusting the background with the negative control. Samples were measured and mean values of the two replicas were presented.
ELISA plates were coated with 100 μL per well of allergens (5 μg mL−1 in PBS) overnight. After washing with TBS–0.05% Tween plates were blocked for 90 min with 1% BSA in TTBS and mice sera (100 μL per well) diluted in TTBS–1% BSA were added (IgE, 1:
3; IgG2a, 1
:
10; IgG2b, 1
:
10, IgG1, 1
:
300; and total IgG, 1
:
300). In parallel, positive anti-Art v 1 serum pool was serially diluted in order to estimate specific antibody arbitrary units, as previously described.14 The plates were incubated with sera in duplicate overnight at 4 °C and washed three times in TTBS. Bound mouse antibodies were detected after 3 h of incubation with 100 μL of biotin-labeled monoclonal anti-mouse IgE, anti IgG1, IgG2a and IgG2b antibodies (BioLegend, San Diego, CA) diluted in TTBS–1% BSA (1 μg mL−1) and alkaline phosphatase-labeled goat anti-mouse IgG diluted 1
:
15
000 (Sigma-Aldrich). After washing with TTBS three times, avidin-AP (Sigma-Aldrich) or AP-labeled anti-goat IgG (Sigma-Aldrich) was added and maintained for 2.5 h. The plates were again washed three times with TTBS and 4-NPP was added as the substrate.
For mass spectrometry analysis, the samples were diluted ten times with a 1% solution of formic acid. Peptides were analyzed on an EASY nLC II system coupled with a LTQ Orbitrap XL (Thermo scientific Inc., MA, USA) previously calibrated with the ProteoMass™ LTQ/FT-Hybrid ESI Positive Mode Cal Mix (MSCAL5 SUPELCO, Sigma Aldrich) calibration set. In total, 240 ng of proteins were injected onto the EASY-Spray PepMap C18 Column (150 × 0.075 mm, 3 μm particle size). Solvent A was a 0.1% formic acid aqueous solution and solvent B was 0.1% formic acid in acetonitrile. A 50 min gradient from 5–70% of solvent B was applied using a 300 nL min−1 flow rate. The CID normalized collision energy value was 35.0%. Parameters for Nanoelectro Spray Ionization (NSI) were as follows: spray voltage +3.91 kV, capillary voltage 6 V, capillary temp 275 °C, tube lens 100V. Spectra were recorded in positive mode in the range of m/z 500–3000. The five most intense monoisotopic peaks in the spectra were fragmented using collision-induced dissociation and measured in the linear ion trap.
Predictions of the peptide sequences were done using the exact masses obtained using MS, exact masses of the native isoforms (Uniprot Q84ZX5) and the peptide calculator tool (http://pepcalc.com/). For the theoretical mass calculations in the peptide calculator, the amino acid sequence for Art v 1 (Uniprot Q84ZX5) was used. To take into account posttranslational modifications, mass additions of 16 Da for the hydroxylation of proline, 2, 4, 6 or 8 Da for partial reduction, as well as 162 Da for galactose and 132 Da for arabinose were used.
The digestion resistant peptides of Art v 1 were modeled in the Modeller software (http://salilab.org/modeller/) based on the NMR solved structure of Art v 1, pdb 2KPY.16
CD spectra were obtained for both proteins with a negative peak at 203 nm (Fig. 1A), which is in accordance to previous findings.17,18 We calculated the % of α-helix, β-sheet, β-turn and random coil (Fig. 1B). The secondary structure content of Art v 1 was virtually unaffected by the modification.
In order to get insight into the tertiary structure changes induced by cis-aconitylation, intrinsic tryptophan fluorescence emission spectra were examined (Fig. 1C). When excited at 280 nm, native Art v 1 exhibited a fluorescence emission maximum (λmax) at 352 nm, originating from the only solvent exposed tryptophan residue (Trp 29) located in the defensine domain. A decrease in intensity was observed due to the quenching of the tryptophan fluorescence by the charged cis-aconityl groups. However, negligible red shifting of the λmax to 353 nm and the fluorescence spectra retaining the overall shape in CAA indicates that cis-aconitylation only slightly disrupted the Art v 1 tertiary structure.
Upon non-covalent binding of the polarity-sensitive hydrophobic fluorescent probe ANS to hydrophobic patches on the protein surfaces, the fluorescence intensity increases and the wavelength of emission maximum demonstrates a blue shift. The decreased ANS binding to CAA with a red shift (from 471 to 475 nm) reflects a higher degree of accessibility for water molecules to the ANS binding region within CAA, indicating that modified Art v 1 has slightly less hydrophobic surfaces (Fig. 1D). These results suggest that cis-aconitylation did not substantially disturb the secondary and tertiary structure of Art v 1, in spite of the fact that Art v 1 was extensively modified by acylation.
IC50 values obtained for the inhibition of human IgE binding to Art v 1 (Fig. 2B) with 13 individual sera from mugwort pollen allergic patients were consistent with the aforementioned result. The IC50 values for Art v 1 and CAA were in the range from 0.001 to 0.656 μg mL−1 (mean = 0.071), and 0.318 to 22.656 μg mL−1, respectively. For all tested sera, the IC50 values increased from 10 to 575-times (mean = 188, n = 13) for CAA compared to Art v 1. This result indicates that the IgE binding epitopes in CAA were significantly altered and cis-aconitylation significantly (p < 0.05) reduced the Art v 1 allergenic potency in the tested group of mugwort-allergic patients.
A significant alteration in the IgE-binding epitopes of CAA also resulted in its reduced ability to degranulate effector cells in the three mugwort pollen-allergic patients. In comparison to Art v 1, a higher concentration of CAA was needed to induce upregulation of CD63 on CD203c+ cells of allergic donors (Fig. 2C). In one of the tested allergic donors (Fig. 2C, #3), CAA did not induce a significant degranulation of basophils, even at the highest concentration that was tested. These results indicate that IgG and IgE epitopes of Art v 1 were significantly modified in CAA which results in a reduced ability to degranulate effector cells.
In response to Art v 1 stimulation, increased amounts of IL-4 (p < 0.05), IL-5 (p < 0.05) and IL-10 (p < 0.01) were detected in donors (Fig. 4B–D). However, there were no statistically significant increases in IL-4, IL-5 and IL-10 production in the cytokine response to CAA, as compared to the unstimulated cultures. In addition, PBMC stimulated with CAA induced less IL-4 (p < 0.05), IL-5 (p < 0.05) and IL-10 (p < 0.01) than native Art v 1. Based on the cytokine profiles, it appears that cis-aconitylated Art v 1 stimulated a Th2-like cell response in PBMC to a much lower extent than Art v 1, although the number of CD3+CD4+ proliferating cells did not differ significantly.
To investigate the potential to block allergen-specific IgE, rabbit IgG antibodies generated to Art v 1 and CAA were tested in an experiment of blocking of the binding of IgE from the serum pool of mugwort pollen allergic patients to the Art v 1-coupled microtiter plate (Fig. 5C). At low sera dilutions (4 and 16), rabbits' anti-CAA and anti-Art v 1 completely blocked human IgE binding to Art v 1. At higher rabbit sera dilutions, anti-CAA exerted a higher blocking capacity (p < 0.05), implying that immunization with CAA resulted in the production of more efficient blocking antibodies of a slightly higher titer.
Our results demonstrate that both Art v 1 and CAA immunization only slightly increased IgE production (Fig. 6A and B). Both Art v 1 forms induced a significant (p < 0.05) increase in total IgG, and there was a tendency of CAA to induce a higher IgG response (Fig. 6C and D). These results demonstrate that the modified Art v 1 maintains the ability to suitably stimulate the immune system to produce specific IgG antibodies directed towards the native Art v 1. Moreover, immunization of mice did not generate significant amount of IgE to CAA.
To get better insight into the differences in IgG responses between Art v 1 and CAA, we have determined the IgG subtypes. Mice immunized with both Art v 1 and CAA demonstrated significantly increased production of IgG1, compared to alum alone (p < 0.005), Fig. 7A and B. However, generation of IgG2a and IgG2b antibodies was not significantly increased in either Art v 1 or CAA-immunized mice (Fig. 7C–F).
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Fig. 8 Endolysosomal in vitro degradation of Art v 1 and CAA during 15 min, 3, 12, 24 and 48 h, using isolated total endolysosomal fractions from murine DCs. (A) Kinetics of endolysosomal degradation of Art v 1 and CAA monitored by SDS PAGE (under reducing conditions), (B) time course of liberation of peptides during endolysosomal degradation of Art v 1 and CAA, (C) remained intact protein (%) during endolysosomal proteolysis of Art v 1 and CAA, calculated from densitometrically quantified protein bands, (D) 3D model of Art v 1 (red), based on NMR structure of Art v 1 (PDB entry 2KPY), overlapped with peptide number 15 identified after 15 min, 3 h and 12 h of endolysosomal degradation of Art v 1 and CAA (blue). |
We analyzed the most dominant high molecular mass peptides by high-resolution mass spectrometry, in order to compare degradation patterns of the native and modified Art v 1. Due to the extremely high diversity of hydrolyzed hydroxyproline-rich domain (Table S1 and Fig. S1 and S2†), it is difficult to map its degradation, but we were able to monitor the degradation of the less variable defensin domain (Fig. 8B, Table S2 and Fig. S3–S12†). After 15 min of endolysosomal processing, the most dominant peptide, with the highest relative intensity, was peptide #15 (5909 Da), originating from and containing the whole defensin domain, which is invariable in all Art v 1 isoforms (Fig. 8B). Its generation in the first minutes of processing suggests that the most proteolysis prone site on Art v 1 is a small linker between defensin and the hydroxyproline-rich domain. Later on, this peptide becomes more and more shortened, and after 48 h the most dominant peptide is 19 (5566 Da), which consists of the whole defensin domain shortened by the N-terminal Lys and C-terminal Cys, suggesting that the defensin domain is highly resistant to proteolytic degradation. Almost the same pattern was observed in Art v 1 and CAA, implying that acid-sensitive modification enabled undisturbed degradation by endolysosomal proteases. In fact, modifications of Lys residues impede recognition and action of enzymes, such as cathepsin S,19 favoring the cleavage of peptide bonds on the carboxyl side of the Lys residues.
The other peptides originating from the defensin domain, as shown in Fig. 8B and summarized in Table S3,† demonstrate that endolysosomal degradation of CAA and Art v 1 results in very similar peptide patterns, suggesting that CAA is capable of providing the same, or a very similar repertoire of MHC class II peptides for binding to T-cell receptors.
It is known that IgE antibodies to Art v 1 are mostly directed against the defensin domain.17,23 By mapping the interactions between Art v 1 and human IgE antibodies, it was shown that IgE interaction sites on Art v 1 are predominantly positively charged.12,16 cis-Aconitylation converts positively charged amino groups into double negative groups, hence resulting in a marked reduction in rabbit IgG and human IgE binding in applied ELISAs and ex vivo basophil degranulation assay. Although the modification did not result in a significant change in protein folding, extensive acylation was efficient in reducing human IgE binding 10 to 575-times in ELISA inhibition with individual human sera.
Further, retaining immunogenicity in animals and the capacity to induce T-cells proliferation of the cis-aconitylated Art v 1 derivative was demonstrated. Both Art v 1 and CAA induced antigen-specific proliferation of PBMCs from mugwort allergic donors. However, in contrast to Art v 1, which induced typical Th2 type cytokines, IL-4, IL-5 and IL-10, T-cell response to the CAA resulted in low cytokine production in PBMCs cultures. Nonprofessional APCs from PBMCs uptake antigens via IgE (e.g. B-cells) and present them to T-cells.24 As CAA has a far lower affinity for IgE than Art v 1, it seems that inefficient uptake and presentation of CAA via B-cells provided a lower secretion of Th2 type cytokines.25,26
The rabbit IgG antibodies, induced by immunization with CAA, were capable of recognizing the native Art v 1 and effectively block human IgE binding to Art v 1. In mice, immunization with CAA led to a similar pattern of humoral immune response. An efficient immunogenicity of CAA may also be due to the more efficient uptake via scavenger receptors (SRs). SRs are expressed on macrophages, B-cells and DCs and recognize diverse polyanionic ligands,27 including many proteins modified by acylation to enhance their negative charge.8,28
It is known that DCs and macrophages are more efficient APCs than B-cells and monocytes of PBMCs, and those APCs from PBMCs are not the actual target APCs for allergen-specific immunotherapy.29 Thereby, in-depth analysis of endolysosomal processing of CAA was undertaken in an in vitro degradome system obtained from dendritic cells. After endolysosomal in vitro degradation, almost the same peptide pattern was observed in Art v 1 and CAA, implying that the applied reversible modification enabled a similar degradation by endolysosomal proteases. It is known that antigens displaying a weak capacity for T-cell priming in vivo were highly susceptible to endolysosomal proteases in vitro.19 Art v 1 harbors only a single immunodominant T-cell epitope (25–36) in the defensin domain and is recognized by 85% of the Art v 1-reactive mugwort pollen allergic patients.30 Epitope mapping of T-cells demonstrated that 14 of 17 patients recognized an Art v 1 epitope in the region 22–36, while five patients recognized an epitope contained in the 43–54 region.31 In our study of the 37 peptides found, region 22–36 was completely retained in 34 identified peptides and region 43–54 in 18 peptides.
Disulfide bonds stabilizing typical α/β folding of the Art v 1 defensin domain32 are arranged with one (C6–C53) external, and three internal disulfides which criss-crossed each other (C17–C37, C22–C47, and C26–C49), resulting in a central defensin region that was more tightly packed and less accessible to proteolytic enzymes. This can explain why we found that most of the degradation resistant peptides are contained in this region and preferably present the immunodominant peptide. Moreover, it was demonstrated that IgE-binding to the Art v 1 single cysteine mutants, C6S and C53S, was almost completely retained,16 while complete loss or only marginal IgE reactivity was observed upon disruption of disulfide bonds C22–C47, C26–C49, or C17–C37, supporting the existence of a tightly packed internal core important for epitope formation, and being hardly accessible to enzyme attack. Under the applied conditions (presence of 2 mM DTT) there was limited reduction, and it appeared that the C6–C53 disulfide was most prone to reduction, which allowed protease to attack up to the internal core, but not the core itself. In the endolysosomal system of APCs, where some of the reductive processes occur,33 the same destiny of the defensin domain could be expected.
In this study, the strategy was focused on the temporary masking of defensin domain epitopes to abrogate IgE binding and also the preservation of the structural motifs necessary for T-cell recognition. Our acid environment-responsive modification aims to deliver epitopes of native Art v 1, by releasing them within a particular site. Therefore this approach can be considered as a “smart” drug delivery system that is able to deliver a therapeutically effective dose in a controlled manner, while minimizing adverse side effects.
There are several advantages to this approach that greatly improve the safety and efficiency of AIT. Firstly, modification results in reduced specific IgE binding, providing reduced allergenic activity and improved safety of allergen vaccine. Secondly, the reversibility of the modification enables the conversion of a modified protein to a native, immunologically fully active, allergen, providing high efficiency and safety. Third, in spite of the reversibility of the modification, the obtained allergen derivative is stable in neutral conditions and it is transformed to the native allergen only in the lysosomal compartment, under the control of intracellular acidification in APCs, where it is degraded.
Preservation of the allergen monomeric molecular size will enable its application in local immunotherapy, in addition to the traditional, subcutaneous application route. Applications of organic acid anhydrides, which are highly reactive and readily transform into organic acids with very low toxicity, further ensure safe preparation.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17261j |
This journal is © The Royal Society of Chemistry 2016 |