Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity but Retained Immunogenicity
© World Allergy Organization; licensee BioMed Central Ltd. 2011
Published: 14 July 2011
Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason.
To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity.
Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant (r) Fel d 1 was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural and recombinant unmodified Fel d 1 were used as controls.
rFel d 1 demonstrated similar IgE binding and biologic activity as its natural counterpart. Upon modification, IgE-binding potency decreased >1000-fold, translating into a >106-fold reduction in biologic activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen.
Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.
It is well established that Fel d 1 is responsible for most IgE reactivity in cat-allergic patients [1–6]. Treatment of cat allergy by immunotherapy with epithelial extracts was demonstrated to be effective [7, 8]. From a regulatory perspective, treatment with mammalian extracts is not ideal because of the potential risk to transmit pathogens. The dominant role of Fel d 1 in cat allergy makes the development of a recombinant Fel d 1-based product an obvious choice. Fel d 1, a 38-kDa glycoprotein with N-linked olisaccharides, was first cloned and expressed by Morgenstern et al . The allergen consists of two 19-kDa noncovalently linked heterodimers, each composed of a light α-chain and a heavy β-chain, encoded by 2 separate genes . Three disulfide bridges connect the α-chain and β-chain in an antiparallel orientation [9, 12]. Expressed as individual chains in Escherichia coli (E. coli), IgE binding was poor,[13, 14] but combining both chains, followed by a lengthy refolding protocol, a recombinant was obtained with similar IgE reactivity as natural Fel d 1 [1, 14, 15]. More recently, Fel d 1 was also expressed as a single construct. In baculovirus both chains were connected in an antiparallel fashion by a linker sequence,[16, 17] whereas in E. coli, both chains were connected in a parallel orientation without linker sequence .
One of the disadvantages of specific immunotherapy is the risk of anaphylactic reactions. By site-directed mutagenesis it is now possible to produce recombinant molecules with reduced IgE binding, so-called hypoallergens. This approach has been reported for several inhalant and food allergens. Mutating 6 amino acids of Bet v 1 gave a hypoallergenic but structurally unchanged molecule [19, 20] and similar results were reported for the peanut allergens Ara h 1-3 [21, 22]. To our knowledge, this strategy has not yet been applied to Fel d 1. The disadvantage of this approach is that it requires considerable insight into the surface structure of an allergen to be able to predict which amino acids should be mutated. Random mutagenesis may be an alternative, but requires laborious screening protocols. Furthermore, reduction of IgE binding is sometimes limited and variable for individual patients [19, 22].
Before the era of molecular biology, chemical modification by, for example, glutaraldehyde or formaldehyde was used as a way to reduce the IgE-binding potency of allergen extracts . This was first described by Marsh et al [24, 25]. Here, we set out to evaluate the feasibility of chemical modification with glutaraldehyde of a recombinant allergen as an alternative to the production of a hypoallergenic mutant obtained by site-directed mutagenesis. To this end, we expressed Fel d 1 as a single construct in a parallel orientation, in E. coli and Pichia pastoris (P. pastoris).
Materials and Methods
Cloning and Expression of Fel d 1
In one construct both chains (α-and β-chain) were cloned together using a linker oligo  in the pPICZαA vector (Invitrogen, San Diego). After transformation into P. pastoris GS115 (His4+) and expression in a Bioflo 3000 bench-top fermentor, cells were harvested and the supernatant was stored at 4°C in 0.1% azide.
A second construct was created by directly linking the C-terminal residue of the β-chain with the N-terminal residue of the α-chain in the pET-19b vector. After transformation into E. coli BL21 (DE3) (Novagen Inc., Madison, WI) and expression, the cell pellet was frozen overnight at -20°C, thawed, and resuspended to one tenth of the original culture volume in 25 mM Tris/2 mM EDTA/pH 7.6. After sonication and pelleting of insoluble matter by centrifugation, the supernatant was saved for further processing of soluble rFel d 1.
Purification of Natural and Recombinant Fel d 1
rFel d 1 was purified in 2 steps: (1) hydrophobic interaction chromatography, using a phenyl-sepharose column; (2) ion-exchange chromatography, using a MonoQ 5/50 GL column (both GE Healthcare Biosciences AB, Uppsala, Sweden). The rFel d 1-containing fractions were pooled, dialyzed against PBS, and concentrated (sixfold), and purity of the sample was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Natural Fel d 1 (nFel d 1) was affinity-purified using monoclonal antibody (mAb) 4F7 as previously described . Protein concentration was measured by the BCA method  (Pierce, Rockford, IL) as described by the manufacturer.
For all experiments E. coli-derived rFel d 1 was used unless indicated otherwise.
Murine Monoclonal Antibodies
MAb 4F7 to nFel d 1 was obtained as described by de Groot et al . MAbs 6B4 and 10F10 against reduced/alkylated nFel d 1 were obtained as described by van Milligen et al . MAb 6B4 had been shown to be specific for the light chain and 10F10 for the heavy chain.
SDS-PAGE and Immunoblotting
SDS-PAGE and immunoblotting were performed as described previously .
Glutaraldehyde Modification of rFel d 1
Glutaraldehyde modification was done similarly for both recombinant preparations. Purified rFel d 1 (5 mL of 1.055 mg/mL from E. coli) was dialyzed on a 1-kDa Amicon membrane with 100 mM sodium carbonate (pH 9.0). The residue was collected and diluted in the same buffer to 21 mL, resulting in a protein concentration of 0.250 mg/mL (13.9 μM, representing a total amount of 292 nmol); 1.37 mL of 25% glutaraldehyde (Merck, Darmstadt, Germany) was slowly added (final amount, 3.26 mmol). As rFel d 1 contains 12 lysine residues and glutaraldehyde has 2 aldehyde groups, the molar ratio aldehyde/rFel d 1 is 1860. The mixture was gently stirred overnight at room temperature; 6.54 mmol of glycine was added in a time span of 30 minutes to quench remaining free aldehyde groups. The mixture was stirred for 1 hour and dialyzed on a 10-kDa Amicon membrane. Protein was not measured in the final product because reliable results cannot be obtained for highly aggregated structures. For further analysis it was assumed that the modification procedure did not result in significant losses. A correction was applied to account for dilution of the sample.
Online High-Performance Size-Exclusion Chromatography Light Scattering
Online high-performance size-exclusion chromatography (HPSEC) light scattering was performed using a 7.8 × 300 mm TSKgel G2000SWXL column (Tosoh Bioscience, Stuttgart, Germany) in single-column mode on a HP1100 analytical chromatography system (Hewlett-Packard, San Jose, CA) equipped with a built-in UV detector and a sequential refractive index (RI), intrinsic viscosity (IV), and rightangle light-scattering detection (TDA 302, Viscotek Corp., Houston, TX) as previously described .
Two female New Zealand white rabbits were immunized and boosted 5 times at 4-week intervals with 100 μg/mL glutaraldehyde-modified rFel d 1 (rFel d 1-mod) from P. pastoris. For each immunization, 1 mL of rFel d 1-mod was mixed with 1 mL of Montanide ISA-50 (Seppic, Paris).
For measurement of specific IgE or IgG against nFel d 1, rFel d 1, and rFel d 1-mod, a radioallergosorbent test (RAST) was performed as described previously . For IgE, the results were expressed as IU/mL, and for IgG, the results were expressed in % bound activity.
ImmunoCAP (Phadia, Uppsala, Sweden) was performed according to the manufacturer's instructions. For CAP-inhibition, a serum pool of cat-allergic patients (n > 100) was preincubated at room temperature for 1 hour with serial dilutions of inhibitor, before addition to cat Immuno-CAP (e1). The following inhibitors were used: nFel d 1, rFel d 1, and rFel d 1-mod. Results were expressed as percentages of inhibition.
To quantify Fel d 1 in different samples, a competitive radioimmunoassay (RIA) or sandwich enzyme-linked immunosorbent assay (ELISA) were used. The competitive RIA was performed essentially as described elsewhere . The Fel d 1 concentration was calculated by using the standard curve of a house dust extract with known Fel d 1 content (14 U/mL). One unit was considered to be equivalent to 1 μg of Fel d 1.
The sandwich ELISA was developed by Chapman et al  (Indoor Biotechnologies, Warminster, UK). Serial sample dilutions were assayed. Reference Fel d 1 standard (9.7 U/mL) was used at serial dilutions from 20 to 0.04 mU/mL. Plates were read in an ELISA microplate reader at 405 nm.
Stripped Basophil Histamine Release Bioassay
To assess biologic activity of Fel d 1, the stripped basophil histamine release (BHR) bioassay was performed. White blood cells were isolated from whole blood of a nonallergic donor by Percoll centrifugation and stripped from IgE by lactic acid treatment as described elsewhere [34, 35]. Subsequently, cells were resensitized with the patient's serum (n = 4). Histamine release was performed with nFel d 1, rFel d 1, and rFel d 1-mod (of 0.01 pg/mL to 100 μg/mL). Liberated histamine was measured by the fluorometric analysis essentially as described by Siraganian .
Allergen-Specific Proliferation of Freshly Isolated Peripheral Blood Mononuclear Cells
T-cell reactivity was assessed by measuring proliferation of peripheral blood mononuclear cells (PBMCs) from cat-allergic donors to nFel d 1, rFel d 1, and rFel d 1-mod. PBMCs were plated out at 2 - 105 cells per well in IMDM (Bio-Whittaker, Walkersville, MD) supplemented with 5% normal human serum (Bio-Whittaker) and gentamycin (80 μg/mL; Duchefa, Haarlem, The Netherlands). Dilution series of allergen or allergoid (0.03-10 μg/mL) were added at day 0, followed by addition of 3H-labeled thymidine at day 6 and harvesting of the radioactively labeled cells at day 7 to measure the Ag-induced proliferation.
Production, Purification, and Modification of rFel d 1
N-Terminal sequencing of the 17 and 20 kDa bands from rFel d 1 (P. pastoris) demonstrated that the lower band is rFel d 1 with the correct N-terminal sequence (EICPAVKRDV) and the upper band is rFel d 1 with 9 residual amino acids originating from the signal peptide, upstream of the Kex 2 site (EEGVSLEKRE). N-Terminal sequencing of the E. coli-derived 17-kDa band showed that the first 10 amino acids were identical to the N-terminal amino acid sequence (β-chain) of Fel d 1 (VKMAETCPIF).
Comparison of Immune Reactivity of Natural and Recombinant Fel d 1
Modified rFel d 1: Reduced IgE and IgG Antibody Binding
Glutaraldehyde-modified rFel d 1 was evaluated by CAP-inhibition, competitive RIA and ELISA. The IgE-binding potency in CAP-inhibition was reduced by >3 orders of magnitude (~1300-fold) compared with that of the unmodified recombinant allergen (Figure 5A). Although the magnitude of reduction observed in competitive RIA (Figure 5C) and ELISA (Figure 5D) was not identical (~500- and ~5000-fold, respectively), it was in both cases highly significant. Similar results were obtained with yeast-derived rFel d 1 (not shown).
Modified rFel d 1: Reduced Biologic Activity
The biologic activity of rFel d 1-mod was assessed by the stripped basophil histamine release bioassay (n = 4). In a first experiment, modified rFel d demonstrated >1000-fold reduced reactivity compared with unmodified rFel d 1, but an accurate assessment was not possible because the native allergen was not diluted far enough (not shown). In a follow-up experiment, for 2 of the 4 original sera, native allergen was diluted up to 0.01 pg/mL, allowing a more accurate calculation. rFel d 1-mod showed a decrease in biologic activity of >106-fold (Figure 5B). Significant reduction (1000-fold) was observed for yeast-derived rFel d 1-mod (not shown). The stripped cells (negative control) showed a release of <4% (not shown).
Modified rFel d 1: Retained T-Cell Proliferation
Immunogenicity of Modified rFel d 1
To assess the capability of rFel d 1-mod to induce IgG antibodies recognizing the natural allergen, 2 rabbits were immunized with the modified allergen from yeast. The resulting rabbit antisera were tested in RAST and immunoblot experiments for IgG reactivity with nFel d 1, rFel d 1, and rFel d 1-mod. IgG antibodies induced upon immunization with rFel d 1-mod bound in a similar fashion to nFel d 1, rFel d 1, and rFel d 1-mod (Figure 5E). Both antisera also clearly detected unmodified rFel d 1 on immunoblot (Figure 4).
In past years, several strategies have been evaluated in our laboratory to express rFel d 1, starting with separate expression of both chains of the molecule in P. pastoris (unpublished data), and subsequently, expression of both chains coupled by a linker sequence (this study). In our hands, this approach resulted in a rFel d 1 preparation with good immune reactivity but an undesirable degree of molecular heterogeneity caused by hyperglycosylation of a fraction of the molecules at the N-linked glycosylation site in the β-chain of Fel d 1. Additionally, some instability of the linker sequence resulted in partial cleavage of the two-chain heterodimer (confirmed by N-terminal sequencing of the resulting fragments). Both post-translational modifications did not significantly affect immune reactivity but the observed heterogeneity is less favorable from a production standpoint. A mutant lacking the N-linked glycosylation site showed decreased heterogeneity, but was still partially cleaved (not shown). Therefore, we chose to express rFel d 1 as a heterodimer of both chains without the instable linker in E. coli resulting in a stable nonglycosylated homogeneous preparation.
Glutaraldehyde modification of this molecule resulted in a truly hypoallergenic molecule with a reduction in IgEbinding potency of around 1000-fold. This translated into a negligible biologic activity (106-fold reduction). To our knowledge this is unmatched by any hypoallergenic strategy using site-directed mutagenesis.
The size distribution in solution demonstrated that modification resulted in a molecular weight ≥1000 kDa. However, under the denaturating conditions of SDS-PAGE, part of the rFel d 1-mod migrated at an apparent molecular weight of 12-20 and 29-40 kDa, indicating that not all aggregation observed in solution was caused by conalent interactions. This suggests that glutaraldehyde modification results in polymerized and aggregated molecules of high MW that may trap also monomers and low MW oligomers, which can be released under reducing and denaturating conditions.
Although the mechanism of allergen-specific immunotherapy is still a matter of debate, it is clear that successful therapy is accompanied by induction of regulatory T-cells and IgG4 antibodies . It is generally accepted that a novel immunotherapy product should have the potency to trigger allergen-specific T-cells. Modified rFel d 1 was shown to induce at least similar T-cell proliferation as its unmodified counterpart. Additionally, a role in immunotherapy of IgG4 as blocking antibodies is likely and we demonstrated that immunization of rabbits with modified rFel d 1 induced specific IgG antibodies that were able to bind to native Fel d 1.
In summary, it was convincingly demonstrated that chemical modification of a recombinant allergen is an easy and highly effective way to achieve hypoallergenicity. This approach will allow safe administration of higher doses of allergen to achieve better efficacy.
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