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Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy
Environmental and occupational respiratory disorders Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy Roberto Gonza´lez-Rioja, PhD,a Ignacio Ibarrola, PhD,a M. Carmen Arilla, PhD,a Angel Ferrer, MD, PhD,b Amparo Mir, MD, PhD,c Carmen Andreu, MD,b Alberto Martı´nez, PhD,a and Juan A. Asturias, PhDa Bilbao, Alicante, and Valencia, Spain
Clinical implications: Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile. (J Allergy Clin Immunol 2007;120:602-9.)
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Background: Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. Objective: The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. Methods: Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. Results: The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. Conclusion: Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy.
Type I allergy, a genetically determined IgE-mediated hypersensitivity, affects almost 25% of the population in developed countries.1 The clinical manifestations of type I allergy can be ameliorated by pharmacotherapy, but allergen-specific immunotherapy (SIT) represents the only curative form of treatment that prevents the progression of the disease.2 Nevertheless, SIT presents the disadvantage of the risk of inducing life-threatening anaphylactic side-effects caused by the administration of active allergens.3 Therefore despite being used in clinical practice since early in the last century, new concepts for successful and safer immunotherapy of type I allergy are greatly required. With the introduction of recombinant DNA technology, it became possible to reduce the allergenic activity of allergens by using recombinant technology.4,5 The current principal approach to allergen alteration is to modify B-cell epitopes to prevent IgE binding and effector cell activation while preserving T-cell epitopes to retain
From athe Research and Development Department, Bial-Arı´stegui, Bilbao; b Servicio de Alergia, Hospital ‘‘Vega Baja’’ de Orihuela, Alicante; and c the Allergy Service, Francisco de Borja Hospital, Gandı´a and Central Research Unit, University of Valencia, Valencia. Supported by Bial-Arı´stegui and by grants FIT-090100-2006-67 from the Programa Nacional de Biomedicina (Accio´n PROFARMA, Ministerio de Industria Turismo y Comercio, Spain) and IT-2005/0000428 from the Programa INNOTEK (Departamento de Industria, Comercio y Turismo, Gobierno Vasco). R.G.-R. is indebted to the Departamento de Industria, Comercio y Turismo and the Departamento de Educacio´n, Universidades e Investigacio´n (Gobierno Vasco) for a predoctoral fellowship. Disclosure of potential conflict of interest: J. A. Asturias has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. A. Martı´nez has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria, Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. I. Ibarrola has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de
Industria, Comercio y Turismo, Gobierno Vasco. M. C. Arilla has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. R. Gonza´lez-Rioja has received support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco, Departamento de Educacio´n, Universidades e Investigacio´n (Gobierno Vasco). The rest of the authors have declared that they have no conflict of interest. The nucleotide sequences for the hybrid proteins Q1, Q2, and Q3 have been deposited in the GenBank database under accession numbers AM490432, AM490433, and AM490434, respectively. Received for publication December 5, 2006; revised April 11, 2007; accepted for publication April 30, 2007. Available online June 13, 2007. Reprint requests: Juan A. Asturias, PhD, Bial-Arı´stegui, R&D, Alameda Urquijo, 27, 48008-Bilbao, Spain. E-mail: [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.04.039
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Key words: Hybrid proteins, IgE epitope, Parietaria pollen allergy, recombinant hypoallergens, T-cell epitope
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the capacity of inducing tolerance.2 B-cell epitopes can be divided structurally into 2 classes: (1) continuous epitopes, in which a stretch of consecutive amino acids spanning 6 to 12 residues are recognized, and (2) discontinuous epitopes, in which residues distant in the primary sequence are brought together by folding of the protein. Native allergens cross-link IgE and induce mast cell and basophil degranulation. They use IgE-facilitated antigen presentation, leading to higher TH2 cytokine production.2 In contrast, modified allergens that lack IgE binding or effector cell degranulation capacities do not use IgE-mediated antigen presentation while using phagocytic or pinocytic antigen uptake mechanisms in dendritic cells, B cells, and macrophages.1,2 This mechanism induces a balanced TH0- or TH1-like cytokine production by T cells and lower IgE and higher IgG production by B cells.6 Pollen allergens are usually low-molecular-weight proteins capable of inducing IgE production by B cells. In particular, Parietaria judaica pollen is the main cause of allergy in the Mediterranean area, with a prevalence of sensitization of 60% to 80% in Italy and Greece and 25% to 40% in Spain and Southern France.7 The composition of allergenic extracts of P judaica pollen has been extensively studied, and it has been observed to contain at least 9 allergens.8 Two major allergens, Par j 1 and Par j 2, have been cloned and sequenced.9,10 Other minor allergens, such as profilin (Par j 3) and calcium-binding protein, have been characterized.11,12 Homology structure comparisons suggest that Par j 1 and Par j 2 belong to the family of proteins referred to as nonspecific lipid transfer proteins.13,14 Continuous and discontinuous IgE epitopes from these allergens together are responsible for almost all of the IgE-binding activity in P judaica pollen.8,12,15,16 Therefore recombinant allergens with known IgE epitopes disrupted by engineered changes to the protein conformation could be used for safer SIT. Here we report the generation of 3 hybrid proteins consisting of fragments of the 2 allergens, Par j 1 and Par j 2, to obtain a vaccine prototype for SIT against P judaica pollen allergy.
METHODS Patients, pollen extract, and natural allergens Thirty patients with P judaica allergy (17 female and 13 male patients; mean age, 30.6 years; age range, 15-58 years) and 15 control subjects (8 female and 7 male subjects; mean age, 45.4 years; age range, 20-75 years) were included in the study. The diagnosis of P judaica allergy was based on clinical history, positive skin prick test (SPT) reactivity, and specific IgE to P judaica pollen extract class 2 or
greater quantified by using the enzyme allergosorbent test (EAST; Hytec-specific IgE EIA; Hycor Biomedical, Kassel, Germany). Control subjects comprised 11 nonatopic subjects and 4 allergic individuals sensitized to different allergenic sources unrelated to P judaica, as demonstrated by negative SPT responses. The use of natural and recombinant allergens in SPTs was approved by the Research Ethics Committee of the Hospital ‘‘Vega Baja’’ de Orihuela, and all patients had given their informed consent. P judaica pollen extract preparation and natural Par j 1 and Par j 2 allergen mix (NPA) purification were performed as previously described.15
Expression and purification of the recombinant hybrid molecules Q1, Q2, and Q3 DNA were generated by using PCR with Par j 1.0103 and Par j 2.0101 (accession nos. AJ969433 and X95865, respectively) as templates (Fig 1). Q1-encoding DNA was amplified with the whole 2 fragments, and Q2-encoding DNA was amplified as 4 fragments, 2 of them corresponding to the Par j 1.0103 sequence (fragment 1, residues 1-28; fragment 2, residues 53-139) and the other 2 corresponding to the Par j 2.0101 sequence (fragment 3, residues 1-28; fragment 4, residues 53-102). Q3-encoding DNA was synthesized by taking the Q2-encoding DNA as the template, and 2 sequences (residues 72-79 from Par j 1.0103 and residues 73-80 from Par j 2.0101) were deleted. Correct sequence and integrity of the open reading frames of all chimeras were confirmed by DNA sequence analysis. Finally, all constructs were cloned in the expression plasmid pQE-32 (Qiagen, Hilden, Germany) and transformed into Escherichia coli M-15. Detailed cloning methods, primer used, and expression and purification conditions are explained in the Methods section in this article’s Online Repository (available, along with Fig E1 and Table E1, at www.jacionline.org).
Electrophoresis and Western blotting SDS-PAGE was performed by using the method of Laemmli under reducing conditions in 14% polyacrylamide gels. Proteins were stained with Coomassie Blue or were detected by means of Western blotting, as described previously,15 by using the serum pool (diluted 1:4) from all of the patients with P judaica allergy included in the study.
Determination of specific IgE levels and inhibition assays Specific IgE levels to P judaica extract and purified proteins were evaluated in duplicate by using EAST with an optimal concentration of protein (50 mg/mL) coupled to cyanogen bromide–activated paper discs. Bound IgE levels were determined with the Hytec specific IgE EIA test (Hycor Biomedical), as described by the manufacturer. For ELISA inhibition, multiwell plates (Greiner, Frickenhausen, Germany) were coated overnight at room temperature with 0.1 mL of P judaica pollen extract (10 mg/mL). The pool of sera (diluted 1:4) from patients with P judaica pollen allergy was preincubated at 48C overnight with solutions containing various concentrations of natural and hybrids proteins. Bound IgE levels were determined as previously described.17
SPTs SPTs were performed with a commercial P judaica pollen extract (12 mg/mL total protein containing 0.7 mg/mL Par j 1–Par j 2; BialArı´stegui, Bilbao, Spain), NPA, and the hybrid molecules (Q1, Q2, and Q3). Sample proteins were diluted in 0.9% NaCl at concentrations of 0.5, 5, and 50 mg/mL, except in the case of the hybrid molecules Q2 and Q3 (5, 50, and 250 mg/mL). Twenty microliters of allergen solution was applied in duplicate on the volar forearm in 2
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Abbreviations used CD: Circular dichroism EAST: Enzyme allergosorbent test NPA: Natural Par j 1-Par j 2 allergen mix SIT: Allergen-specific immunotherapy SPT: Skin prick test
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FIG 1. Schematic construction of hybrid proteins. Identified T-cell epitope regions and putative IgE-binding epitope regions are marked by yellow and green, respectively. Numbering indicates amino acid position, and primers are indicated by arrows. Disulfide bridges are indicated by straight lines at the top of the scheme. Dashed lines show deleted stretches.
opposite directions with sterile prick lancets. The sizes of the elicited wheals were determined by means of computerized planimetry (AutoCAD 11; Autodesk, Inc, San Rafael, Calif). Wheal surface areas of greater than 7 mm2 were considered positive.
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PBMCs were isolated from 13 patients with P judaica allergy by means of density gradient centrifugation on lymphocyte separation solution (Lymphoprep; Nycomed, Oslo, Norway), and their proliferation was assayed as previously described.18 Briefly, microcultures of 2 3 105 PBMCs in a final volume of 200 mL of medium (AIM-V serum-free Medium; Gibco, Paisley, United Kingdom) were performed in flat-bottom microplates (Nunclon; NUNC, Roskilde, Denmark). For each sample, triplicate wells were incubated (at 378C and humidified atmosphere of 5% CO2 in air) with P judaica extract and purified proteins at final protein concentrations of 0.5, 5, 50, 500, and 5000 ng/mL. In all cases, triplicate unstimulated cultures were included. After 3 days, cell proliferation was quantified by using a colorimetric method, according to the manufacturer’s instructions (Cell Proliferation Reagent WST-1; Roche Diagnostics, Barcelona, Spain). The percentage of proliferation was calculated as follows: ðS2CÞ=C 3 100, where S and C represent the absorbance values for antigen-stimulated and control cells, respectively.
Circular dichroism Far-UV (190-250 nm) circular dichroism (CD) spectra at pH 7.0 and 208C were recorded with a Jasco J-810 spectropolarimeter (Jasco Europe, Cremella, Italy). Protein concentration was 0.035 mg/mL in 20 mmol/L sodium phosphate buffer in a 0.2-cm cuvette, and 40 scans were accumulated.
Statistical analysis Statistical data analysis was performed by using the Wilcoxon test and the Spearman r correlation included in the SPSS 11.0 for Windows software package (SPSS, Inc, Chicago, Ill). A P value of less than .05 was considered statistically significant.
RESULTS Characteristics of the recombinant hybrid molecules We generated 3 hybrid proteins using the 2 major allergens of P judaica pollen, Par j 1 and Par j 2. The first
FIG 2. A, Coomassie-stained SDS-PAGE and IgE Western blotting of NPA (lane 1), Q1 (lane 2), Q2 (lane 3), and Q3 (lane 4) incubated with the serum pool of patients with P judaica allergy. The arrowhead indicates the IgE-reacting band of Q2. B, CD spectra of NPA and the hybrid molecules.
one, called Q1, is composed of the whole sequences of both major allergens, resulting in a cDNA construct with Par j 1 at the N-terminus and Par j 2 at the C-terminus. This protein was expressed as a 32-kd His-tagged fusion protein (Fig 2), with a final yield of 2 mg/L bacterial culture. The other 2 hybrid proteins are deletion mutants of Q1: Q2 lacks the stretches from 29 to 52 residues in
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Reduced in vitro IgE binding of recombinant hybrid proteins We further determined whether the 3 hybrid proteins exhibited reduced binding of serum-specific IgE from patients with P judaica pollen allergy in comparison with native allergens. Western blotting with a pool of sera from allergic patients showed only IgE binding to NPA and Q1 (Fig 2, A). The deletions introduced in Q2 and Q3 affected drastically the IgE-binding capacity of these proteins. In the case of Q2, a very weak recognition of IgE antibodies could be observed, whereas it was totally absent in Q3 (Fig 2, A). The capacity of human IgE to bind the hybrid was also tested by using EAST with individual sera from allergic patients (Fig 3, A). Q1 (median, 10.7 IU/mL; lower and upper 95% confidence limits of 5.4 and 33.4 IU/mL, respectively) showed lower IgE-binding capacity than NPA (median, 13.2 IU/mL; 95% confidence limit, 8.455.5 IU/mL). In the case of the deletion mutants Q2 (median, 1.2 IU/mL; 95% confidence limits, 0.7-1.9 IU/mL) and Q3 (median, 0.4 IU/mL; 95% confidence limits, 0.30.5 IU/mL), serum specific IgE from all tested individuals showed almost no binding activity compared with NPA (P < .001). When comparing with the whole extract (median, 8.9 IU/mL; 95% confidence limits, 5.1-18.6 IU/mL), 29 of 30 patients sera showed lower IgE reactivity to Q2 and Q3 (P < .001). For complete information of individual patients, see Table E2 in this article’s Online Repository at www.jacionline.org. Further analysis of IgE reactivity was done by means of ELISA inhibition with the pool of sera, confirming the reduced ability of the 3 hybrid proteins to inhibit IgE binding to the whole extract in patient sera (Fig 3, B). An 80-fold higher concentration of Q1 than NPA was necessary to reach 50% of IgE-binding activity inhibition to the P judaica pollen extract. In the case of Q2 and Q3, a more than 1500-fold higher concentration had to be used to obtain the same inhibition. In no case did hybrid proteins reach the IgE inhibition obtained by NPA, which was able to inhibit almost 95% of the IgE-binding capacity of the serum pool.
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both components, whereas in Q3 an additional short stretch of 8 residues (72-79 in Par j 1 and 73-80 in Par j 2) was deleted. Q3 also has an amino acid exchange from PCR cloning: Gln-1/Pro. Q2 and Q3 proteins were expressed as 28-kd His-tagged fusion proteins with a final yield of 5 and 7 mg/L culture, respectively (Fig 2, A). Secondary structure elements were analyzed by using CD spectroscopy, applying NPA and the hybrid proteins. The spectra of the hybrid proteins were nearly identical among them but totally different from the natural allergens’ CD spectra. NPA spectra presented a minimum at 208 nm, a well-defined shoulder at 222 nm, and a maximum at 190 nm, whereas spectra of the hybrid proteins shifted toward random coil conformation, reaching an almost completely unfolded state (Fig 2, B).
FIG 3. A, Box plot representation of duplicate specific IgE means from individual patient sera (n 5 30) to P judaica pollen extract, NPA, Q1, Q2, and Q3. Outliers are indicated by open circles. B, ELISA inhibition curves. The binding of human IgE from the serum pool to P judaica pollen extract was inhibited by using NPA and Q1, Q2, and Q3 hybrid proteins. Values represent the mean 1 SD (n 5 3).
Reduced in vivo allergenic activity of recombinant hybrid proteins The ability of the native allergens Par j 1 and Par j 2 and the fusion proteins to elicit cutaneous reactions in vivo was evaluated in 30 patients with P judaica allergy (Fig 4, A). The whole group of patients had positive skin reactions to P judaica extract and to 5 and 50 mg/mL NPA. On the contrary, at 5 mg/mL, only 5 patients had a positive SPT response to Q1, and none of them occurred when deletion mutants were used. When the highest concentration was used in the assay (50 mg/mL for Q1 and 250 mg/mL for Q2 and Q3), it was be observed that whereas 26 of 30
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FIG 4. A, SPT results from individual patients (n 5 30) with P judaica pollen extract: NPA and Q1 (both at 50 mg/ mL) and Q2 and Q3 (both at 250 mg/mL) are shown. B, Induction of T-cell proliferation. PBMCs from individual patients (n 5 13) were stimulated with 500 ng/mL P judaica pollen extract, NPA, Q1, Q2, and Q3. Individual values are given as means of duplicate wheal surface areas and of triplicate stimulation index (SI) percentages, respectively.
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patients had positive SPT responses in the case of Q1, only 6 and 4 patients reacted with Q2 and Q3, respectively. Nevertheless, in all positive responses elicited by the deletion mutants, we observed a dramatically reduced skin test reactivity compared with NPA at 50 mg/mL (median, 104.5 mm2; 95% confidence limits, 79.3-121.5 mm2) and the whole extract (median, 61.8 mm2; 95% confidence limits, 55.9-69.5 mm2; P < .001; Fig 4, A). No significant difference (P 5 .195) was found between skin reactivity of NPA at 0.5 mg/mL (median, 32.5 mm2; 95% confidence limits, 27.5-37.5 mm2) and Q1 at 50 mg/mL (median, 37.5 mm2; 95% confidence limits, 29.9-45.5 mm2), showing that Q1 was about 100-fold less reactive than NPA. No positive reaction was observed in control subjects, either to the extract or to the purified allergens, indicating the high specificity of the test. No adverse side effects were observed in all the subjects tested.
T-cell reactivity of recombinant hybrid proteins To investigate the T cell–stimulating capacity of the fusion proteins, specific proliferation of PBMCs from 13 patients with P judaica pollen allergy was analyzed by using pollen extract and purified proteins at final protein concentrations of 0.5, 5, 50, 500, and 5000 ng/mL. Purified proteins followed a dose–response curve, and comparative analyses were performed at maximum response (500 ng/mL). The results showed that Q1 and Q2 induced a T-cell proliferation not significantly different from that obtained with the natural allergens (P 5 .484 and P 5 .182, respectively) and the extract (P 5 .152 and P 5 .294, respectively; Fig 4, B). On the other hand, Q3 induced significantly lower proliferation compared with the extract (P 5 .002) and NPA (P 5 .004; Fig 4, B).
DISCUSSION The present study describes the construction of 3 hybrid molecules consisting of the 2 major allergens of the P judaica pollen, Par j 1 and Par j 2, to obtain a preventive allergy vaccine with reduced activity for IgE binding but retaining T-cell reactivity. The aim of this study was to define hypoallergenic molecules that might be used in the future as alternative vaccines for a successful immunotherapy, replacing the commonly used whole natural allergen extracts. There are several major disadvantages of using native allergen extracts in a preventive allergy vaccine.19-21 Extracts are usually poorly characterized because they contain complex mixtures of varying amounts of both allergens and other nonallergenic components.19 For this reason, highly heterogeneous immune responses have been observed in patients undergoing specific immunotherapy based on allergen extracts.22-24 Furthermore, proteases from mites, fungus, and P judaica pollen extracts have been involved in the degradation of bioactive peptides,25 and therefore the use of extracts containing these enzymes can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs.25 Nevertheless, with the application of recombinant DNA technology in the field of allergen characterization, pure recombinant allergens have become available for the formulation of defined and safer allergy vaccines,4,26 and the use of hybrid technology can overcome the problems of low representation and poor immunogenicity of certain allergens, as well as simplifying production and registration procedures.27 We chose the P judaica pollen allergy model because it is basically a 2-component allergenic system,10 and NPA is able to inhibit up to 95% of the total IgE-binding activity of the P judaica pollen extract (Fig 3, B), which suggests that allergens not related to Par j 1 and Par j 2 are
remarkably less important from a clinical point of view. Minor and cross-reactive allergens, profilin (Par j 3) and a calcium-binding protein, have also been described,11,12 but previous experiments performed in our laboratory showed that none of 30 sera reacted against any of these minor allergens (data not shown). These results are in agreement with the low incidence of sensitization of Par j 3 and pellitory calcium-binding protein (3% and 6%, respectively) found in the Italian population.12 Although it has been described that Par j 1 and Par j 2 share similar IgE epitopes14 and Par j 2 could be used as marker for pellitory pollen allergy,28,29 inclusion of Par j 1 and Par j 2 in a single hybrid molecule would extend the T-cell repertoire and induce strong protective antibody responses, as described for grass pollen hybrids.30,31 The rationale for the construction of the first hybrid protein, Q1, is based on the conformational dependence of B-cell epitopes.32 In E coli folding of cysteine-containing proteins is difficult because of the reducing state of the bacterial cytoplasm, and therefore expression of these proteins results frequently in the accumulation of protein aggregates.33 After solubilization, the refolding step is also complex because of the formation of random intramolecular and intermolecular disulfide bonds.34 Considering that Q1 contains 16 cysteine residues, it could be predicted that the proportion of native-like folded protein would be drastically lower than that of its separate native components. For investigation of the IgE reactivity, we used 4 different methods: Western blotting, EAST, ELISA inhibition, and SPT. Q1 retained some capacity to bind IgE antibodies, as can be observed in Western blot and EAST experiments, but to a lower extent than the extract and NPA. These results are in concordance with the ELISA inhibition results, in which Q1 showed an 80-fold reduction when compared with natural Par j 1–Par j 2 for reaching the 50% of inhibition of IgE binding activity in P judaica pollen extract. This reduction of in vitro IgE reactivity was also shown by a decrease of Q1 cutaneous reactivity, which presented a positive response at the highest concentration used in SPTs (50 mg/mL), being quite lower than that elicited by NPA (about 100-fold). Different conformational hypoallergenic variants of Par j 1 alone have been previously obtained by means of disruption of disulfide bridges with site-directed mutagenesis.16 Loss of IgE-binding activity is a common feature of fusion proteins independent of their construction, such as fusion of the same allergen (Bet v 1 trimers),35 homologous proteins (vespid venom allergens),36 or unrelated proteins (different bee venom allergens).34 One exception is grass pollen allergen hybrids, which contained most of the B-cell epitopes of the individual components.30,31 Surprisingly, most of the hybrid molecules described conserved a considerable amount of secondary structure.30,31,35,36 The analysis of the secondary structure of the hybrid molecule Q1 by means of CD showed that it had lost most of the secondary structure present in NPA, suggesting the destruction of the conformational B-cell epitopes present in the 2 allergens. Some linear epitopes
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not affected by structural changes could be responsible for the residual IgE-binding activity found in this hybrid molecule (Fig 3). Epitope mapping studies of Par j 1 and Par j 2 allergens has revealed the presence of several possible IgE-binding epitopes in both allergens.13,14,37 On the basis of these studies, we generated another 2 hybrid molecules, namely Q2 and Q3. In Q2 the fragments from residues 29 to 52 in Par j 1 and Par j 2, containing 4 cysteines involved in the 4 disulfide bridges, were deleted (Fig 1). In this manner there was no possibility of forming any disulfide bridge, and most of the possible continuous B epitopes were removed.14 This hybrid molecule showed a complete reduction of in vitro IgE reactivity, with a great decrease in IgE binding, as assessed by means of Western blotting and EAST, when compared with Q1. ELISA inhibition studies showed a greater than 100-fold reduction of IgE binding when compared with Q1 and a greater than 1500-fold reduction with regard to NPA. In vivo activity of Q2 showed no positive reaction at the same concentration observed by Q1 (50 mg/mL), and only a slight reaction in 6 of 30 patients was detected at a concentration of 250 mg/mL. Hybrid Q3 displayed 2 additional deletions of 8 residues with respect to Q2, corresponding to 2 putative continuous IgE epitopes. In this case the results obtained in in vitro and in vivo assays were almost identical to those showed by Q2. Because the secondary structure of hybrid proteins Q1, Q2, and Q3 reached an almost completely unfolded state, the previously reported continuous IgE epitopes (1-II, 1-III, 2-III, 2-IV, and 2-V) in Par j 1 and Par j 214 might be of some importance, as has been reported for one allergen of Phleum pratense pollen, Phl p 5b.38 Nevertheless, it cannot be ruled out that this reduced allergenicity could be also due to the destruction of conformational IgE epitopes located in different parts of the molecules rather than due to the loss of continuous epitopes in the deleted regions themselves. Very recently, a fusion protein of Par j 2–Par j 1 composed of conformational hypoallergenic variants obtained by means of disulfide bridge disruption with site-directed mutagenesis has been reported to elicit 100- to 1000-fold less allergenicity than the wild-type allergens.39 On the other hand, T lymphocytes are crucial cellular components in the induction of IgE, as well as in the downregulation of IgE responses during SIT.1,6 Intact T-cell epitopes are required to enable the induction of specific T-cell tolerance or anergy against the allergen.2 Because only one immunodominant T-cell epitope has been identified in Par j 1 to date,40 T-cell reactivity of these hybrid molecules was analyzed by means of stimulation of PBMCs to ensure that the majority of the Par j 1 and Par j 2 T-cell repertoire was retained. As can be expected, Q1, composed of the complete sequences of Par j 1 and Par j 2, was able to induce T-cell proliferation to an extent similar to the whole extract and NPA. Q2 had a similar effect. On the contrary, recombinant Q3 showed much lower allergen-specific T-lymphocyte activation, suggesting that the deletion of the sequences has disrupted putative T-cell epitopes.
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Very recently, the first results from immunotherapy trials with recombinant allergens41 or recombinant hypoallergenic derivatives42,43 have begun to become available. In general, these studies demonstrated that such treatment was safe, induced IgG1 response, and reduced IgE levels against corresponding natural allergens. In conclusion, our results suggest that the Q2 allergen derivative could be a good candidate for successful immunotherapy against P judaica pollen allergy and might be preferable to the whole extract–based treatment. We thank the Servicio de Secuenciacio´n (Centro de Investigaciones Biolo´gicas-CSIC, Madrid, Spain) for DNA-sequencing facilities and Dr A. R. Viguera (Unidad de Biofı´sica, Universidad del Paı´s Vasco-CSIC, Leioa, Spain) for analysis of CD spectra. REFERENCES
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1. Akdis CA, Blaser K. Regulation of specific immune responses by chemical and structural modifications of allergens. Int Arch Allergy Immunol 2000;121:261-9. 2. Akdis CA, Blaser K. Mechanisms of allergen-specific immunotherapy. Allergy 2000;55:518-24. 3. Westritschnig K, Valenta R. Can we genetically engineer safer and more effective immunotherapy reagents? Curr Opin Allergy Clin Immunol 2003;3:495-500. 4. Valenta R, Linhart B. Molecular design of allergy vaccines. Curr Opin Immunol 2005;17:1-10. 5. Ferreira F, Briza P, Infu¨hr D, Schmidt G, Wallner M, Wopfner N, et al. Modified recombinant allergens for safer immunotherapy. Inflamm Allergy Drug Targets 2006;5:5-14. 6. Akdis CA, Blase´ K. Regulation of specific immune responses by chemical and structural modifications of allergens. Int Arch Allergy Immunol 2000;121:261-9. 7. Colombo P, Duro G, Costa MA, Izzo V, Mirisola M, Locorotondo G, et al. An update on allergens. Parietaria pollen allergens. Allergy 1998;53:917-21. 8. Colombo P, Bonura A, Costa M, Izzo V, Passantino R, Licorotondo G, et al. The allergens of Parietaria. Int Arch Allergy Immunol 2003;130:173-9. 9. Costa MA, Colombo P, Izzo V, Kennedy H, Venturella S, Cocchiara R, et al. cDNA cloning expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen. FEBS Lett 1994;341:182-6. 10. Duro G, Colombo P, Costa MA, Izzo V, Porcasi R, DiFiore R, et al. cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen. FEBS Lett 1996;399:295-8. 11. Asturias JA, Ibarrola I, Eseverri JL, Arilla MC, Go´nzalez-Rioja R, Martı´nez A. PCR-based cloning and immunological characterization of Parietaria judaica pollen profilin. J Invest Allergol Clin Immunol 2004;14:43-8. 12. Trapani A, Gulino L, Bonura A, Artale A, Geraci D, Di Felice G, et al. Cross-reactive allergens of the Parietaria judaica pollen. In: Abstract Book for the XXV EAACI Congress; Vienna, Austria; June 10-14, 2006. p. 15. 13. Colombo P, Kennedy D, Ramsdale T, Costa MA, Duro G, Izzo V, et al. Identification of an immunodominant IgE epitope of the Parietaria judaica major allergen. J Immunol 1998;160:2780-5. 14. Asturias JA, Go´mez-Bayo´n N, Eseverri JL, Martı´nez A. Par j 1 and Par j 2, the major allergens from Parietaria judaica pollen, have similar immunoglobulin E epitopes. Clin Exp Allergy 2003;33:518-24. 15. Arilla MC, Gonza´lez-Rioja R, Ibarrola I, Mir A, Monteseirı´n J, Conde J, et al. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2. Clin Exp Allergy 2006;36:87-93. 16. Bonura A, Amoroso S, Locorotondo G, Di Felice G, Tinghino R, Geraci D, et al. Hypoallergenic variants of the Parietaria judaica major allergen Par j 1: a member of the non-specific lipid transfer protein plant family. Int Arch Allergy Immunol 2001;126:32-40. 17. Asturias JA, Ibarrola I, Eraso E, Arilla MC, Martı´nez A. The major Platanus acerifolia pollen allergen Pla a 1 has sequence homology to invertase inhibitors. Clin Exp Allergy 2003;33:978-85.
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18. Ibarrola I, Sanz ML, Gamboa PM, Mir A, Benahmed D, Ferrer A, et al. Biological Characterization of glutaraldehyde-modified Parietaria judaica pollen extracts. Clin Exp Allergy 2004;34:303-9. 19. Bousquet J, Lockey R, Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases. Allergy 1998;53:1-42. 20. van der Veen MJ, Mulder M, Witteman AM, van Ree R, Aalberse RC, Jansen HM, et al. False-positive skin prick test responses to commercially available dog dander extracts caused by contamination with house dust mite (Dermatophagoides pteronyssinus) allergens. J Allergy Clin Immunol 1996;98:1028-34. 21. Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003;111:777-83. 22. Mothes N, Heinzkill M, Drachenberg KJ. Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvant vaccine: reduced seasonally boosted IgE production and inhibition of basophil histamine release by blocking antibodies. Clin Exp Allergy 2003;33:1198-208. 23. Moverate R, Elfman L, Vesterinen E, Metso T, Haahtela T. Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System. Allergy 2002;57:423-30. 24. Ball T, Sperr WR, Valent P. Introduction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy. Eur J Immunol 1999;29:2026-36. 25. Cortes L, Carvalho AL, Todo-Bom A, Faro C, Pires E, Verı´ssimo P. Purification of a novel aminopeptidase from the pollen of Parietaria judaica that alters epithelial integrity and degrades neuropeptides. J Allergy Clin Immunol 2006;118:878-84. 26. Valenta R, Kraft D. Recombinant allergens: from production and characterization to diagnosis, treatment, and prevention of allergy. Methods 2004;32:207-8. 27. Linhart B, Valenta R. Vaccine engineering improved by hybrid technology. Int Arch Allergy Immunol 2004;134:324-31. 28. Stumvoll S, Westritschnig K, Lidholm J, Spitzauer S, Colombo P, Duro G, et al. Identification of cross-reactive and genuine Parietaria judaica pollen allergens. J Allergy Clin Immunol 2003;111:974-9. 29. Gonza´lez-Rioja R, Ferrer A, Arilla MC, Ibarrola I, Viguera AR, Andreu C, et al. Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens. Clin Exp Allergy 2007;37:243-50. 30. Linhart B, Jahn-Schmid B, Verdino P, Keller W, Ebner C, Kraft D, et al. Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity. FASEB J 2002;16:1301-3. 31. Linhart B, Hartl A, Jahn-Schmid B, Verdino P, Keller W, Valenta R. A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination. J Allergy Clin Immunol 2005;115:1010-6. 32. Aalberse RC. Structural biology of allergens. J Allergy Clin Immunol 2000;106:228-38. 33. Georgiou G, Valax P. Expression of correctly folded proteins in Escherichia coli. Curr Opin Biotechnol 1996;7:190-7. 34. Kussebi F, Karamloo F, Rhyner C, Schmid-Grendelmeier P, Salagianni M, Mannhart C, et al. A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy. J Allergy Clin Immunol 2005;115:323-9. 35. Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi F, Akdis CA, et al. Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen, Bet v 1. FASEB J 2001;15:2045-7. 36. King TP, Jim SY, Monsalve RI, Kagey-Sobotka A, Lichtenstein LM, Spangfort MD. Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s. J Immunol 2001;166: 6057-65. 37. Costa MA, Duro G, Izzo V, Colombo P, Mirisola MG, Locorotondo G, et al. The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogenously recognized among allergic subjects. Allergy 2000;55:246-50. 38. Schramm G, Kahlert H, Suck R, Weber B, Stuwe HT, Muller WD, et al. ‘‘Allergen engineering’’: variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity. J Immunol 1999;162:2406-14. 39. Bonura A, Corinti S, Artale A, Di Felice G, Amoroso S, Melis M, et al. A hybrid expressing genetically engineered major allergens of the Parietaria pollen as a tool for specific allergy vaccination. Int Arch Allergy Immunol 2007;142:274-84.
40. De Palma R, Wu S, Sallusto F, Di Felice G, Martucci P, Geraci D, et al. Use of antagonist peptides to inhibit in vitro T cell responses to Par j 1, the major allergen of Parietaria judaica pollen. J Immunol 1999;162:1982-7. 41. Jutel M, Jaeger L, Suck R, Meyer H, Fiebig H, Cromwell O. Allergenspecific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 2005;116:608-13.
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42. Niederberger V, Horak F, Vrtala S, Spitzauer S, Krauth MT, Valent P, et al. Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Natl Acad Sci U S A 2004;101:14677-82. 43. Cromwell O, Fiebig H, Suck R, Kahlert H, Nandy A, Kettner J, et al. Strategies for recombinant allergen vaccines and fruitful results from first clinical studies. Immunol Allergy Clin North Am 2006;26:261-81.
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Clinical implications: Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile. (J Allergy Clin Immunol 2007;120:602-9.)
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Background: Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. Objective: The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. Methods: Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. Results: The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. Conclusion: Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy.
Type I allergy, a genetically determined IgE-mediated hypersensitivity, affects almost 25% of the population in developed countries.1 The clinical manifestations of type I allergy can be ameliorated by pharmacotherapy, but allergen-specific immunotherapy (SIT) represents the only curative form of treatment that prevents the progression of the disease.2 Nevertheless, SIT presents the disadvantage of the risk of inducing life-threatening anaphylactic side-effects caused by the administration of active allergens.3 Therefore despite being used in clinical practice since early in the last century, new concepts for successful and safer immunotherapy of type I allergy are greatly required. With the introduction of recombinant DNA technology, it became possible to reduce the allergenic activity of allergens by using recombinant technology.4,5 The current principal approach to allergen alteration is to modify B-cell epitopes to prevent IgE binding and effector cell activation while preserving T-cell epitopes to retain
From athe Research and Development Department, Bial-Arı´stegui, Bilbao; b Servicio de Alergia, Hospital ‘‘Vega Baja’’ de Orihuela, Alicante; and c the Allergy Service, Francisco de Borja Hospital, Gandı´a and Central Research Unit, University of Valencia, Valencia. Supported by Bial-Arı´stegui and by grants FIT-090100-2006-67 from the Programa Nacional de Biomedicina (Accio´n PROFARMA, Ministerio de Industria Turismo y Comercio, Spain) and IT-2005/0000428 from the Programa INNOTEK (Departamento de Industria, Comercio y Turismo, Gobierno Vasco). R.G.-R. is indebted to the Departamento de Industria, Comercio y Turismo and the Departamento de Educacio´n, Universidades e Investigacio´n (Gobierno Vasco) for a predoctoral fellowship. Disclosure of potential conflict of interest: J. A. Asturias has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. A. Martı´nez has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria, Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. I. Ibarrola has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de
Industria, Comercio y Turismo, Gobierno Vasco. M. C. Arilla has received grant support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco. R. Gonza´lez-Rioja has received support from Programa Nacional de Biomedicina, Ministerio de Industria Turismo y Comercio, Spain, and from the Programa INNOTEK, Departamento de Industria, Comercio y Turismo, Gobierno Vasco, Departamento de Educacio´n, Universidades e Investigacio´n (Gobierno Vasco). The rest of the authors have declared that they have no conflict of interest. The nucleotide sequences for the hybrid proteins Q1, Q2, and Q3 have been deposited in the GenBank database under accession numbers AM490432, AM490433, and AM490434, respectively. Received for publication December 5, 2006; revised April 11, 2007; accepted for publication April 30, 2007. Available online June 13, 2007. Reprint requests: Juan A. Asturias, PhD, Bial-Arı´stegui, R&D, Alameda Urquijo, 27, 48008-Bilbao, Spain. E-mail: [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.04.039
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Key words: Hybrid proteins, IgE epitope, Parietaria pollen allergy, recombinant hypoallergens, T-cell epitope
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the capacity of inducing tolerance.2 B-cell epitopes can be divided structurally into 2 classes: (1) continuous epitopes, in which a stretch of consecutive amino acids spanning 6 to 12 residues are recognized, and (2) discontinuous epitopes, in which residues distant in the primary sequence are brought together by folding of the protein. Native allergens cross-link IgE and induce mast cell and basophil degranulation. They use IgE-facilitated antigen presentation, leading to higher TH2 cytokine production.2 In contrast, modified allergens that lack IgE binding or effector cell degranulation capacities do not use IgE-mediated antigen presentation while using phagocytic or pinocytic antigen uptake mechanisms in dendritic cells, B cells, and macrophages.1,2 This mechanism induces a balanced TH0- or TH1-like cytokine production by T cells and lower IgE and higher IgG production by B cells.6 Pollen allergens are usually low-molecular-weight proteins capable of inducing IgE production by B cells. In particular, Parietaria judaica pollen is the main cause of allergy in the Mediterranean area, with a prevalence of sensitization of 60% to 80% in Italy and Greece and 25% to 40% in Spain and Southern France.7 The composition of allergenic extracts of P judaica pollen has been extensively studied, and it has been observed to contain at least 9 allergens.8 Two major allergens, Par j 1 and Par j 2, have been cloned and sequenced.9,10 Other minor allergens, such as profilin (Par j 3) and calcium-binding protein, have been characterized.11,12 Homology structure comparisons suggest that Par j 1 and Par j 2 belong to the family of proteins referred to as nonspecific lipid transfer proteins.13,14 Continuous and discontinuous IgE epitopes from these allergens together are responsible for almost all of the IgE-binding activity in P judaica pollen.8,12,15,16 Therefore recombinant allergens with known IgE epitopes disrupted by engineered changes to the protein conformation could be used for safer SIT. Here we report the generation of 3 hybrid proteins consisting of fragments of the 2 allergens, Par j 1 and Par j 2, to obtain a vaccine prototype for SIT against P judaica pollen allergy.
METHODS Patients, pollen extract, and natural allergens Thirty patients with P judaica allergy (17 female and 13 male patients; mean age, 30.6 years; age range, 15-58 years) and 15 control subjects (8 female and 7 male subjects; mean age, 45.4 years; age range, 20-75 years) were included in the study. The diagnosis of P judaica allergy was based on clinical history, positive skin prick test (SPT) reactivity, and specific IgE to P judaica pollen extract class 2 or
greater quantified by using the enzyme allergosorbent test (EAST; Hytec-specific IgE EIA; Hycor Biomedical, Kassel, Germany). Control subjects comprised 11 nonatopic subjects and 4 allergic individuals sensitized to different allergenic sources unrelated to P judaica, as demonstrated by negative SPT responses. The use of natural and recombinant allergens in SPTs was approved by the Research Ethics Committee of the Hospital ‘‘Vega Baja’’ de Orihuela, and all patients had given their informed consent. P judaica pollen extract preparation and natural Par j 1 and Par j 2 allergen mix (NPA) purification were performed as previously described.15
Expression and purification of the recombinant hybrid molecules Q1, Q2, and Q3 DNA were generated by using PCR with Par j 1.0103 and Par j 2.0101 (accession nos. AJ969433 and X95865, respectively) as templates (Fig 1). Q1-encoding DNA was amplified with the whole 2 fragments, and Q2-encoding DNA was amplified as 4 fragments, 2 of them corresponding to the Par j 1.0103 sequence (fragment 1, residues 1-28; fragment 2, residues 53-139) and the other 2 corresponding to the Par j 2.0101 sequence (fragment 3, residues 1-28; fragment 4, residues 53-102). Q3-encoding DNA was synthesized by taking the Q2-encoding DNA as the template, and 2 sequences (residues 72-79 from Par j 1.0103 and residues 73-80 from Par j 2.0101) were deleted. Correct sequence and integrity of the open reading frames of all chimeras were confirmed by DNA sequence analysis. Finally, all constructs were cloned in the expression plasmid pQE-32 (Qiagen, Hilden, Germany) and transformed into Escherichia coli M-15. Detailed cloning methods, primer used, and expression and purification conditions are explained in the Methods section in this article’s Online Repository (available, along with Fig E1 and Table E1, at www.jacionline.org).
Electrophoresis and Western blotting SDS-PAGE was performed by using the method of Laemmli under reducing conditions in 14% polyacrylamide gels. Proteins were stained with Coomassie Blue or were detected by means of Western blotting, as described previously,15 by using the serum pool (diluted 1:4) from all of the patients with P judaica allergy included in the study.
Determination of specific IgE levels and inhibition assays Specific IgE levels to P judaica extract and purified proteins were evaluated in duplicate by using EAST with an optimal concentration of protein (50 mg/mL) coupled to cyanogen bromide–activated paper discs. Bound IgE levels were determined with the Hytec specific IgE EIA test (Hycor Biomedical), as described by the manufacturer. For ELISA inhibition, multiwell plates (Greiner, Frickenhausen, Germany) were coated overnight at room temperature with 0.1 mL of P judaica pollen extract (10 mg/mL). The pool of sera (diluted 1:4) from patients with P judaica pollen allergy was preincubated at 48C overnight with solutions containing various concentrations of natural and hybrids proteins. Bound IgE levels were determined as previously described.17
SPTs SPTs were performed with a commercial P judaica pollen extract (12 mg/mL total protein containing 0.7 mg/mL Par j 1–Par j 2; BialArı´stegui, Bilbao, Spain), NPA, and the hybrid molecules (Q1, Q2, and Q3). Sample proteins were diluted in 0.9% NaCl at concentrations of 0.5, 5, and 50 mg/mL, except in the case of the hybrid molecules Q2 and Q3 (5, 50, and 250 mg/mL). Twenty microliters of allergen solution was applied in duplicate on the volar forearm in 2
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Abbreviations used CD: Circular dichroism EAST: Enzyme allergosorbent test NPA: Natural Par j 1-Par j 2 allergen mix SIT: Allergen-specific immunotherapy SPT: Skin prick test
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FIG 1. Schematic construction of hybrid proteins. Identified T-cell epitope regions and putative IgE-binding epitope regions are marked by yellow and green, respectively. Numbering indicates amino acid position, and primers are indicated by arrows. Disulfide bridges are indicated by straight lines at the top of the scheme. Dashed lines show deleted stretches.
opposite directions with sterile prick lancets. The sizes of the elicited wheals were determined by means of computerized planimetry (AutoCAD 11; Autodesk, Inc, San Rafael, Calif). Wheal surface areas of greater than 7 mm2 were considered positive.
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PBMCs were isolated from 13 patients with P judaica allergy by means of density gradient centrifugation on lymphocyte separation solution (Lymphoprep; Nycomed, Oslo, Norway), and their proliferation was assayed as previously described.18 Briefly, microcultures of 2 3 105 PBMCs in a final volume of 200 mL of medium (AIM-V serum-free Medium; Gibco, Paisley, United Kingdom) were performed in flat-bottom microplates (Nunclon; NUNC, Roskilde, Denmark). For each sample, triplicate wells were incubated (at 378C and humidified atmosphere of 5% CO2 in air) with P judaica extract and purified proteins at final protein concentrations of 0.5, 5, 50, 500, and 5000 ng/mL. In all cases, triplicate unstimulated cultures were included. After 3 days, cell proliferation was quantified by using a colorimetric method, according to the manufacturer’s instructions (Cell Proliferation Reagent WST-1; Roche Diagnostics, Barcelona, Spain). The percentage of proliferation was calculated as follows: ðS2CÞ=C 3 100, where S and C represent the absorbance values for antigen-stimulated and control cells, respectively.
Circular dichroism Far-UV (190-250 nm) circular dichroism (CD) spectra at pH 7.0 and 208C were recorded with a Jasco J-810 spectropolarimeter (Jasco Europe, Cremella, Italy). Protein concentration was 0.035 mg/mL in 20 mmol/L sodium phosphate buffer in a 0.2-cm cuvette, and 40 scans were accumulated.
Statistical analysis Statistical data analysis was performed by using the Wilcoxon test and the Spearman r correlation included in the SPSS 11.0 for Windows software package (SPSS, Inc, Chicago, Ill). A P value of less than .05 was considered statistically significant.
RESULTS Characteristics of the recombinant hybrid molecules We generated 3 hybrid proteins using the 2 major allergens of P judaica pollen, Par j 1 and Par j 2. The first
FIG 2. A, Coomassie-stained SDS-PAGE and IgE Western blotting of NPA (lane 1), Q1 (lane 2), Q2 (lane 3), and Q3 (lane 4) incubated with the serum pool of patients with P judaica allergy. The arrowhead indicates the IgE-reacting band of Q2. B, CD spectra of NPA and the hybrid molecules.
one, called Q1, is composed of the whole sequences of both major allergens, resulting in a cDNA construct with Par j 1 at the N-terminus and Par j 2 at the C-terminus. This protein was expressed as a 32-kd His-tagged fusion protein (Fig 2), with a final yield of 2 mg/L bacterial culture. The other 2 hybrid proteins are deletion mutants of Q1: Q2 lacks the stretches from 29 to 52 residues in
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Reduced in vitro IgE binding of recombinant hybrid proteins We further determined whether the 3 hybrid proteins exhibited reduced binding of serum-specific IgE from patients with P judaica pollen allergy in comparison with native allergens. Western blotting with a pool of sera from allergic patients showed only IgE binding to NPA and Q1 (Fig 2, A). The deletions introduced in Q2 and Q3 affected drastically the IgE-binding capacity of these proteins. In the case of Q2, a very weak recognition of IgE antibodies could be observed, whereas it was totally absent in Q3 (Fig 2, A). The capacity of human IgE to bind the hybrid was also tested by using EAST with individual sera from allergic patients (Fig 3, A). Q1 (median, 10.7 IU/mL; lower and upper 95% confidence limits of 5.4 and 33.4 IU/mL, respectively) showed lower IgE-binding capacity than NPA (median, 13.2 IU/mL; 95% confidence limit, 8.455.5 IU/mL). In the case of the deletion mutants Q2 (median, 1.2 IU/mL; 95% confidence limits, 0.7-1.9 IU/mL) and Q3 (median, 0.4 IU/mL; 95% confidence limits, 0.30.5 IU/mL), serum specific IgE from all tested individuals showed almost no binding activity compared with NPA (P < .001). When comparing with the whole extract (median, 8.9 IU/mL; 95% confidence limits, 5.1-18.6 IU/mL), 29 of 30 patients sera showed lower IgE reactivity to Q2 and Q3 (P < .001). For complete information of individual patients, see Table E2 in this article’s Online Repository at www.jacionline.org. Further analysis of IgE reactivity was done by means of ELISA inhibition with the pool of sera, confirming the reduced ability of the 3 hybrid proteins to inhibit IgE binding to the whole extract in patient sera (Fig 3, B). An 80-fold higher concentration of Q1 than NPA was necessary to reach 50% of IgE-binding activity inhibition to the P judaica pollen extract. In the case of Q2 and Q3, a more than 1500-fold higher concentration had to be used to obtain the same inhibition. In no case did hybrid proteins reach the IgE inhibition obtained by NPA, which was able to inhibit almost 95% of the IgE-binding capacity of the serum pool.
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both components, whereas in Q3 an additional short stretch of 8 residues (72-79 in Par j 1 and 73-80 in Par j 2) was deleted. Q3 also has an amino acid exchange from PCR cloning: Gln-1/Pro. Q2 and Q3 proteins were expressed as 28-kd His-tagged fusion proteins with a final yield of 5 and 7 mg/L culture, respectively (Fig 2, A). Secondary structure elements were analyzed by using CD spectroscopy, applying NPA and the hybrid proteins. The spectra of the hybrid proteins were nearly identical among them but totally different from the natural allergens’ CD spectra. NPA spectra presented a minimum at 208 nm, a well-defined shoulder at 222 nm, and a maximum at 190 nm, whereas spectra of the hybrid proteins shifted toward random coil conformation, reaching an almost completely unfolded state (Fig 2, B).
FIG 3. A, Box plot representation of duplicate specific IgE means from individual patient sera (n 5 30) to P judaica pollen extract, NPA, Q1, Q2, and Q3. Outliers are indicated by open circles. B, ELISA inhibition curves. The binding of human IgE from the serum pool to P judaica pollen extract was inhibited by using NPA and Q1, Q2, and Q3 hybrid proteins. Values represent the mean 1 SD (n 5 3).
Reduced in vivo allergenic activity of recombinant hybrid proteins The ability of the native allergens Par j 1 and Par j 2 and the fusion proteins to elicit cutaneous reactions in vivo was evaluated in 30 patients with P judaica allergy (Fig 4, A). The whole group of patients had positive skin reactions to P judaica extract and to 5 and 50 mg/mL NPA. On the contrary, at 5 mg/mL, only 5 patients had a positive SPT response to Q1, and none of them occurred when deletion mutants were used. When the highest concentration was used in the assay (50 mg/mL for Q1 and 250 mg/mL for Q2 and Q3), it was be observed that whereas 26 of 30
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FIG 4. A, SPT results from individual patients (n 5 30) with P judaica pollen extract: NPA and Q1 (both at 50 mg/ mL) and Q2 and Q3 (both at 250 mg/mL) are shown. B, Induction of T-cell proliferation. PBMCs from individual patients (n 5 13) were stimulated with 500 ng/mL P judaica pollen extract, NPA, Q1, Q2, and Q3. Individual values are given as means of duplicate wheal surface areas and of triplicate stimulation index (SI) percentages, respectively.
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patients had positive SPT responses in the case of Q1, only 6 and 4 patients reacted with Q2 and Q3, respectively. Nevertheless, in all positive responses elicited by the deletion mutants, we observed a dramatically reduced skin test reactivity compared with NPA at 50 mg/mL (median, 104.5 mm2; 95% confidence limits, 79.3-121.5 mm2) and the whole extract (median, 61.8 mm2; 95% confidence limits, 55.9-69.5 mm2; P < .001; Fig 4, A). No significant difference (P 5 .195) was found between skin reactivity of NPA at 0.5 mg/mL (median, 32.5 mm2; 95% confidence limits, 27.5-37.5 mm2) and Q1 at 50 mg/mL (median, 37.5 mm2; 95% confidence limits, 29.9-45.5 mm2), showing that Q1 was about 100-fold less reactive than NPA. No positive reaction was observed in control subjects, either to the extract or to the purified allergens, indicating the high specificity of the test. No adverse side effects were observed in all the subjects tested.
T-cell reactivity of recombinant hybrid proteins To investigate the T cell–stimulating capacity of the fusion proteins, specific proliferation of PBMCs from 13 patients with P judaica pollen allergy was analyzed by using pollen extract and purified proteins at final protein concentrations of 0.5, 5, 50, 500, and 5000 ng/mL. Purified proteins followed a dose–response curve, and comparative analyses were performed at maximum response (500 ng/mL). The results showed that Q1 and Q2 induced a T-cell proliferation not significantly different from that obtained with the natural allergens (P 5 .484 and P 5 .182, respectively) and the extract (P 5 .152 and P 5 .294, respectively; Fig 4, B). On the other hand, Q3 induced significantly lower proliferation compared with the extract (P 5 .002) and NPA (P 5 .004; Fig 4, B).
DISCUSSION The present study describes the construction of 3 hybrid molecules consisting of the 2 major allergens of the P judaica pollen, Par j 1 and Par j 2, to obtain a preventive allergy vaccine with reduced activity for IgE binding but retaining T-cell reactivity. The aim of this study was to define hypoallergenic molecules that might be used in the future as alternative vaccines for a successful immunotherapy, replacing the commonly used whole natural allergen extracts. There are several major disadvantages of using native allergen extracts in a preventive allergy vaccine.19-21 Extracts are usually poorly characterized because they contain complex mixtures of varying amounts of both allergens and other nonallergenic components.19 For this reason, highly heterogeneous immune responses have been observed in patients undergoing specific immunotherapy based on allergen extracts.22-24 Furthermore, proteases from mites, fungus, and P judaica pollen extracts have been involved in the degradation of bioactive peptides,25 and therefore the use of extracts containing these enzymes can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs.25 Nevertheless, with the application of recombinant DNA technology in the field of allergen characterization, pure recombinant allergens have become available for the formulation of defined and safer allergy vaccines,4,26 and the use of hybrid technology can overcome the problems of low representation and poor immunogenicity of certain allergens, as well as simplifying production and registration procedures.27 We chose the P judaica pollen allergy model because it is basically a 2-component allergenic system,10 and NPA is able to inhibit up to 95% of the total IgE-binding activity of the P judaica pollen extract (Fig 3, B), which suggests that allergens not related to Par j 1 and Par j 2 are
remarkably less important from a clinical point of view. Minor and cross-reactive allergens, profilin (Par j 3) and a calcium-binding protein, have also been described,11,12 but previous experiments performed in our laboratory showed that none of 30 sera reacted against any of these minor allergens (data not shown). These results are in agreement with the low incidence of sensitization of Par j 3 and pellitory calcium-binding protein (3% and 6%, respectively) found in the Italian population.12 Although it has been described that Par j 1 and Par j 2 share similar IgE epitopes14 and Par j 2 could be used as marker for pellitory pollen allergy,28,29 inclusion of Par j 1 and Par j 2 in a single hybrid molecule would extend the T-cell repertoire and induce strong protective antibody responses, as described for grass pollen hybrids.30,31 The rationale for the construction of the first hybrid protein, Q1, is based on the conformational dependence of B-cell epitopes.32 In E coli folding of cysteine-containing proteins is difficult because of the reducing state of the bacterial cytoplasm, and therefore expression of these proteins results frequently in the accumulation of protein aggregates.33 After solubilization, the refolding step is also complex because of the formation of random intramolecular and intermolecular disulfide bonds.34 Considering that Q1 contains 16 cysteine residues, it could be predicted that the proportion of native-like folded protein would be drastically lower than that of its separate native components. For investigation of the IgE reactivity, we used 4 different methods: Western blotting, EAST, ELISA inhibition, and SPT. Q1 retained some capacity to bind IgE antibodies, as can be observed in Western blot and EAST experiments, but to a lower extent than the extract and NPA. These results are in concordance with the ELISA inhibition results, in which Q1 showed an 80-fold reduction when compared with natural Par j 1–Par j 2 for reaching the 50% of inhibition of IgE binding activity in P judaica pollen extract. This reduction of in vitro IgE reactivity was also shown by a decrease of Q1 cutaneous reactivity, which presented a positive response at the highest concentration used in SPTs (50 mg/mL), being quite lower than that elicited by NPA (about 100-fold). Different conformational hypoallergenic variants of Par j 1 alone have been previously obtained by means of disruption of disulfide bridges with site-directed mutagenesis.16 Loss of IgE-binding activity is a common feature of fusion proteins independent of their construction, such as fusion of the same allergen (Bet v 1 trimers),35 homologous proteins (vespid venom allergens),36 or unrelated proteins (different bee venom allergens).34 One exception is grass pollen allergen hybrids, which contained most of the B-cell epitopes of the individual components.30,31 Surprisingly, most of the hybrid molecules described conserved a considerable amount of secondary structure.30,31,35,36 The analysis of the secondary structure of the hybrid molecule Q1 by means of CD showed that it had lost most of the secondary structure present in NPA, suggesting the destruction of the conformational B-cell epitopes present in the 2 allergens. Some linear epitopes
Gonza´lez-Rioja et al 607
not affected by structural changes could be responsible for the residual IgE-binding activity found in this hybrid molecule (Fig 3). Epitope mapping studies of Par j 1 and Par j 2 allergens has revealed the presence of several possible IgE-binding epitopes in both allergens.13,14,37 On the basis of these studies, we generated another 2 hybrid molecules, namely Q2 and Q3. In Q2 the fragments from residues 29 to 52 in Par j 1 and Par j 2, containing 4 cysteines involved in the 4 disulfide bridges, were deleted (Fig 1). In this manner there was no possibility of forming any disulfide bridge, and most of the possible continuous B epitopes were removed.14 This hybrid molecule showed a complete reduction of in vitro IgE reactivity, with a great decrease in IgE binding, as assessed by means of Western blotting and EAST, when compared with Q1. ELISA inhibition studies showed a greater than 100-fold reduction of IgE binding when compared with Q1 and a greater than 1500-fold reduction with regard to NPA. In vivo activity of Q2 showed no positive reaction at the same concentration observed by Q1 (50 mg/mL), and only a slight reaction in 6 of 30 patients was detected at a concentration of 250 mg/mL. Hybrid Q3 displayed 2 additional deletions of 8 residues with respect to Q2, corresponding to 2 putative continuous IgE epitopes. In this case the results obtained in in vitro and in vivo assays were almost identical to those showed by Q2. Because the secondary structure of hybrid proteins Q1, Q2, and Q3 reached an almost completely unfolded state, the previously reported continuous IgE epitopes (1-II, 1-III, 2-III, 2-IV, and 2-V) in Par j 1 and Par j 214 might be of some importance, as has been reported for one allergen of Phleum pratense pollen, Phl p 5b.38 Nevertheless, it cannot be ruled out that this reduced allergenicity could be also due to the destruction of conformational IgE epitopes located in different parts of the molecules rather than due to the loss of continuous epitopes in the deleted regions themselves. Very recently, a fusion protein of Par j 2–Par j 1 composed of conformational hypoallergenic variants obtained by means of disulfide bridge disruption with site-directed mutagenesis has been reported to elicit 100- to 1000-fold less allergenicity than the wild-type allergens.39 On the other hand, T lymphocytes are crucial cellular components in the induction of IgE, as well as in the downregulation of IgE responses during SIT.1,6 Intact T-cell epitopes are required to enable the induction of specific T-cell tolerance or anergy against the allergen.2 Because only one immunodominant T-cell epitope has been identified in Par j 1 to date,40 T-cell reactivity of these hybrid molecules was analyzed by means of stimulation of PBMCs to ensure that the majority of the Par j 1 and Par j 2 T-cell repertoire was retained. As can be expected, Q1, composed of the complete sequences of Par j 1 and Par j 2, was able to induce T-cell proliferation to an extent similar to the whole extract and NPA. Q2 had a similar effect. On the contrary, recombinant Q3 showed much lower allergen-specific T-lymphocyte activation, suggesting that the deletion of the sequences has disrupted putative T-cell epitopes.
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Very recently, the first results from immunotherapy trials with recombinant allergens41 or recombinant hypoallergenic derivatives42,43 have begun to become available. In general, these studies demonstrated that such treatment was safe, induced IgG1 response, and reduced IgE levels against corresponding natural allergens. In conclusion, our results suggest that the Q2 allergen derivative could be a good candidate for successful immunotherapy against P judaica pollen allergy and might be preferable to the whole extract–based treatment. We thank the Servicio de Secuenciacio´n (Centro de Investigaciones Biolo´gicas-CSIC, Madrid, Spain) for DNA-sequencing facilities and Dr A. R. Viguera (Unidad de Biofı´sica, Universidad del Paı´s Vasco-CSIC, Leioa, Spain) for analysis of CD spectra. REFERENCES
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