Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor

To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed. Mutants with one or more cysteine residues altered were constructed with a histidine residue tag for purification purposes and expressed as recombinant proteins in E. coli. Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72 77) and one mutant with all 6 cysteines altered (6 Cys). The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding. Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2. However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered. A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72 77 or the 6 Cys mutant. Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 microg/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved. The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy.     

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.     

IgG1, IgG4 and IgE antibody reactivity to mutant forms of the major dust mite allergen Lep d 2 among atopic and nonatopic subjects naturally exposed to Lepidoglyphus destructor

BACKGROUND: Lepidoglyphus destructor is a common dust mite causing IgE-mediated sensitization. The major allergen, Lep d 2, has previously been cloned and expressed as several double Cys to Ser mutants with the purpose of producing hypoallergenic variants for immunotherapy. Our aim was to investigate the reactivity pattern of IgG1, IgG4 and IgE antibodies to wild-type (wt) rLep d 2 and four mutants among atopic and nonatopic subjects in relation to sensitization and exposure to L. destructor. METHODS: Inhibition and sandwich ELISA were used to compare IgG1, IgG4 and IgE antibody reactivities to rLep d 2 variants in serum of 20 atopic and 18 nonatopic farmers naturally exposed to L. destructor. A group of 22 urban subjects served as controls. RESULTS: Atopic farmers demonstrated correlating IgE and IgG4 levels to rLep d 2(wt) (rs = 0.70; p < 0.0001) which were significantly (p < 0.0001) higher than those of nonatopic farmers and urban controls. No IgG4 antibodies were detected in nonatopic farmers despite chronic allergen exposure. A parallel reactivity pattern of IgE and IgG4 to all rLep d 2 mutants was observed. The mutant lacking all 3 disulfide bonds, rLep d 2.6Cys, demonstrated neither any IgE nor IgG4 reactivity. In contrast, IgG1 antibodies had a different reactivity pattern and were detected among most subjects irrespective of atopy, exposure to L. destructor or disulfide impairments in rLep d 2. Moreover, IgG1 levels to rLep d 2(wt) and rLep d 2.6Cys correlated (n = 60; rs = 0.65; p < 0.0001). CONCLUSIONS: IgE/IgG4 Ab to rLep d 2 were restricted to atopic farmers and demonstrated parallel recognition patterns of conformational epitopes. In contrast, IgG1 antibodies are ubiquitously found and mainly recognize sequential structures. The observed isotypic difference and interindividual variation in antibody specificities among atopic and nonatopic subjects imply careful investigation of hypoallergenic variants destined for immunotherapy.     

T cell responses to recombinant isoforms, synthetic peptides and a mutant variant of Lep d 2, a major allergen from the dust mite Lepidoglyphus destructor

BACKGROUND: The dust mite Lepidoglyphus destructor is an important cause of allergic reactions to dust, especially in farming environments. Two isoforms, recombinant (r)Lep d 2.01 and rLep d 2.02, of the major allergen Lep d 2, have previously been expressed as recombinant proteins. These isoforms differ 10.4% at the amino acid level. Furthermore, a mutant form of Lep d 2.01 (rLep d 2.6Cys) with a highly reduced IgE reactivity, has also been produced. OBJECTIVE: To investigate the T cell responses to the recombinant isoforms of Lep d 2, the Lep d 2.6Cys mutant and peptides of Lep d 2, in allergic and non-allergic individuals. METHODS: Peripheral blood mononuclear cells from 18 allergic and 16 non-allergic individuals were stimulated with the different antigens and the proliferative responses were measured. The cytokine production (interleukin (IL)-4, IL-5 and interferon (IFN)-gamma) were measured by ELISA. RESULTS: Higher T cell proliferation was measured to isoform 01 than to 02 in 28/34 subjects. The responses to rLep d 2.6Cys were lower than to isoform 01 in most subjects, but higher than to Lep d 2.02. Two immuno-dominant peptides, corresponding to amino acid residue 11-25 and 61-75 were identified. The atopic subjects produced significantly lower IFN-gamma in response to Lep d 2.01 as compared to the non-atopics. CONCLUSIONS: There was a significant difference in T cell response between the two isoforms of rLep d 2. The hypoallergenic mutant rLep d 2.6Cys was able to evoke a T cell response with a magnitude which is between the two isoforms. Amino acid residue 11-25 and 61-75 are the most frequently recognized parts of Lep d 2 and are likely to contain the immuno-dominant T cell epitopes.     

An ELISA for recombinant Lepidoglyphus destructor, Lep d 2, and the monitoring of exposure to dust mite allergens in farming households

BACKGROUND: Exposure to indoor allergens, such as dust mites, has been recognized as a risk factor for sensitization and symptoms. OBJECTIVE: To develop a two-site ELISA for the determination of Lep d 2 in the reservoir, to measure dust mite allergen exposure (Lep d 2, Der p 1, Der f 1 and Der 2) in farm households, and to investigate whether exposure to these allergens is associated with sensitization, asthma and rhinoconjunctivitis. METHODS: Monoclonal antibodies to recombinant (r)Lep d 2 were produced with standard hybridoma technique. Dust samples from 393 households were analysed for allergen content by two-site ELISA methods. RESULTS: A two-site Lep d 2 ELISA was developed with a detection limit of 0.09 microg/g. The assay was highly reproducible and levels of Lep d 2 showed a strong correlation with the number of Lepidoglyphus mites (r(s): 0.7; P = 0.0002). Lep d 2 was detected in 20% of the homes; levels ranged from 0.09 to 1.7 microg/g of dust. Der p 1 was recorded in 59% of the samples, ranging from 0.055 to 139 microg/g, and Der f 1 and Der 2 in 40% and 50% of the samples, ranging from 0.055 to 24.5 microg/g and 24.3 microg/g, respectively. Dermatophagoides allergens were significantly higher in mattresses than in carpets (P < 0.0001), but this difference was not observed with Lep d 2. A strong relationship between immunoglobulin (Ig)E to rLep d 2 and asthma (OR = 10.4) and rhinoconjunctivitis (OR = 7.5) was seen. Furthermore, sensitization to D. pteronyssinus was significantly associated with asthma (OR: 13.7) and rhinoconjunctivitis (OR: 5.7). CONCLUSION: When assessing mite allergen exposure in rural homes, not only the Der p 1, Der f 1 and Der 2 allergens, but also the Lep d 2 allergen should be taken into consideration.     

Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus, Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae with recombinant allergens

BACKGROUND: Dust mites are important inducers of allergic disease. Group 2 allergens are recognized as major allergens in several mite species, including Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae. No allergens have thus far been characterized on the molecular level from the dust mite Glycyphagus domesticus. OBJECTIVE: We sought to examine the cross-reactivity among group 2 allergens of G domesticus, L destructor, T putrescentiae, and D pteronyssinus. METHODS: A group 2 allergen from G domesticus, Gly d 2, was cloned and expressed as a recombinant protein. Cross-reactivity between Gly d 2 and 3 other group 2 allergens, Lep d 2, Tyr p 2, and Der p 2, was studied by using individual sera and a serum pool RAST-positive to G domesticus, L destructor, T putrescentiae, and D pteronyssinus. Recombinant allergens were used as inhibitors of IgE binding in immunoblotting experiments. Molecular modeling on the basis of the Der p 2 structure was carried out for Gly d 2, Lep d 2, and Tyr p 2. RESULTS: Two cDNAs encoding isoforms of Gly d 2 were isolated, but only the Gly d 2.02 isoform was used in this study. Sixteen of 17 subjects had IgE to Gly d 2. The protein sequence of Gly d 2 revealed 79% identity to Lep d 2 and 46% and 41% identity to Tyr p 2 and Der p 2, respectively. Extensive cross-reactivity was demonstrated among Gly d 2, Lep d 2, and Tyr p 2, but little cross-reactivity was found between these allergens and Der p 2. According to the tertiary structure of Der p 2 and 3-dimensional models of Gly d 2, Lep d 2, and Tyr p 2, differences reside mainly in surface-exposed residues. CONCLUSION: Gly d 2 showed high sequence homology to Lep d 2. Cross-reactivity was observed between Gly d 2, Lep d 2, and Tyr p 2, but only limited cross-reactivity was demonstrated between these 3 allergens and Der p 2.     

The proteolytic activity of the major dust mite allergen Der p 1 enhances the IgE antibody response to a bystander antigen

BACKGROUND: We have recently demonstrated that immunization of mice with proteolytically active Der p 1, the major dust mite allergen, results in a significant enhancement in total and Der p 1-specific IgE synthesis compared to mice immunized with Der p 1 that has been irreversibly blocked with the cysteine protease inhibitors E-64 and iodoacetamide. Thus, the demonstration that the proteolytic activity of Der p 1 enhances total IgE production, apart from increasing Der p 1-specific IgE, suggests that this allergen may have an IgE-specific adjuvant effect. OBJECTIVE: To determine if the proteolytic activity of Der p 1 has an IgE-specific adjuvant effect. METHODS: We have examined this concept in experiments whereby ovalbumin, used as a bystander antigen, was injected alone or coinjected with either proteolytically active or inactive Der p 1 into groups of mice and IgE and IgG antibody responses were measured. RESULTS: Here we demonstrate for the first time that the proteolytic activity of Der p 1, when given at 10-fold higher concentration, enhances the IgE antibody response to ovalbumin. CONCLUSIONS: These findings show that the proteolytic activity of Der p 1 leads to the augmentation of IgE antibody responses to itself and to other allergens present in the microenvironment.     

Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the Pharmacia CAP system.

BACKGROUND: Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE-mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts Lepidoglyphus destructor and Tyrophagus putrescentiae in the Pharmacia RAST CAP System. METHODS: The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. RESULTS: The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial L. destructor and T. putrescentiae assays. Two subjects out of 416, who tested negative in the commercial L. destructor assay, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the T. putrescentiae extract were 5/418. The possibility that these subjects were sensitised to L. destructor and T. putrescentiae could not be excluded. CONCLUSIONS: The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of L. destructor and T. putrescentiae. It appears likely that the addition of just a few more recombinant L. destructor and T. putrescentiae allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to L. destructor and T. putrescentiae.     

Prevention of allergen-specific IgE production and suppression of an established Th2-type response by immunization with DNA encoding hypoallergenic allergen derivatives of Bet v 1, the major birch-pollen allergen

In atopic patients, programming towards a preferential Th2 immunity leads to IgE antibody production and cellular Th2 immunity against otherwise harmless antigens. We report the development of prophylactic and therapeutic DNA vaccines for the major birch-pollen allergen, Bet v 1. We constructed three DNA vaccines, coding for the complete cDNA, coding for two hypoallergenic fragments or coding for a hypoallergenic Bet v 1 mutant. The protective effect was studied in mice pretreated by intradermal DNA injections, then sensitized with Bet v 1 protein. Mice pretreated with any of the three Bet v 1-specific DNA vaccines were protected against allergic sensitization to Bet v 1. Protection was characterized by a lack of Bet v 1-specific IgE production, a lack of basophil activation and an enhanced IFN-gamma expression. DNA vaccines with wild-type Bet v 1 induced strong Bet v 1-specific antibody responses whereas DNA vaccines with hypoallergenic Bet v 1 derivatives induced no (fragments) or only transient (mutant) Bet v 1-specific antibody responses. A therapeutic approach with the fragment-DNA vaccine reduced IgE production and stimulated a sustained Th1 cytokine milieu. Our results demonstrate that DNA vaccines with hypoallergenic forms of the allergen specifically protect against sensitization and suppress established Th2-type responses. This concept may be applied for the development of safe and specific DNA vaccines for the prophylaxis and therapy of allergic diseases.     

H2 genotype and IgE immune response to Fel dI, the cat major allergen, in mice.

Mice of 6 strains were immunized with a highly purified Fel dI allergen adsorbed to alum. Their ability to display a significant IgE response was detected via passive cutaneous anaphylaxis (PCA) tests, performed in rats. Linkage of the responsiveness to the H2 genotype is not totally obvious. IgE response can be delayed, particularly in the SJL strain (H2-s histocompatibility allele), and lacking in the C57 B1/6 strain (H2-b). Mice with H2-k, H2-d alleles and the B6D2F1 hybrid H2-b/d are good responders, leading to the hypothesis that the IgE response to Fel dI may be related to the H2-d allele. For all good responders, individual variations are rather important. All our observations show that mice perfectly mimic human hypersensitization to the cat major allergen Fel dI, at least where the IgE response is concerned.     

The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

Characterization of IgE epitopes of Cuc m 2, the major melon allergen,and their role in cross‐reactivity with pollen profilins

Background Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross‐reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes. Objective To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross‐reaction among members of this family. Methods IgE of melon‐allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2‐specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three‐dimensional surfaces of other profilins. Results Seventeen melon‐allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass‐, and birch‐pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S₂W₃A₅Y₆D₉H₁₀T₁₁₁P₁₁₂G₁₁₃Q₁₁₄N₁₁₆M₁₁₇R₁₂₁L₁₂₂. The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. Conclusions The identified mimotope could be involved in cross‐reactions among food and pollen profilins. Many of these cross‐reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies. Cite this as: L. Tordesillas, L. F. Pacios, A. Palacín, J. Cuesta‐Herranz, M. Madero and A. Díaz‐Perales, Clinical & Experimental Allergy, 2010 (40) 174–181.     

A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus.

The potent allergenicity of Der p 1, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is thought to be related to its cysteine protease activity. Therefore, there is considerable interest in developing a sensitive assay for measuring Der p 1 activity to screen for specific inhibitors. This study demonstrates for the first time that the activity of Der p 1 can be measured conveniently in a continuous rate assay with the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (K(m) = 280 microM and kcat/K(m) = 4.6 x 10(3)/M/s).     

Differences between specificities of IgE and IgG4 antibodies: studies using recombinant chain 1 and chain 2 of the major cat allergen Felis domesticus (Fel d) I

IgE- and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE- and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE- and IgG4 binding was found. The fraction of Pel d I-specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.     

A sensitive reverse ELISA for the measurement of specific IgE to Der p 2, a major Dermatophagoides pteronyssinus allergen.

BACKGROUND: Epidemiologic studies have shown that the presence of IgE antibodies to house dust mite and other indoor allergens is an important risk factor for asthma. OBJECTIVE: The aim of this study was to develop a reverse ELISA (rELISA) for measuring specific IgE to Der p 2, a major Dermatophagoides pteronyssinus (Dpt) allergen, as a potential tool for followup of allergen immunotherapy. METHODS: Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 was used to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Sera from 48 asthmatic patients with positive skin prick test (SPT+) to D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results were compared with a group of 41 SPT asthmatic and 30 SPT- control subjects. RESULTS: The sensitivity of the two assays for Der p 2-specific IgE was 3.9 EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISA and rELISA (r = 0.74; P < 0.0001). However, rELISA was more sensitive than was cELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Der p 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT+ asthmatic patients. CONCLUSIONS: rELISA has shown to be a sensitive and alternative method for measuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cell epitopes in allergens will provide valuable information for the design of allergen analogs and peptides for immunotherapy.     

A chimeric human-cat Fcgamma-Fel d1 fusion protein inhibits systemic, pulmonary, and cutaneous allergic reactivity to intratracheal challenge in mice sensitized to Fel d1, the major cat allergen

Co-aggregation of FcepsilonRI with FcgammaRIIb can block FcepsilonRI-mediated reactivity and Fc gamma:allergen chimeric proteins, by co-crosslinking FcgammaRIIb to allergen-specific IgE bound to the FcepsilonRI can block allergen-specific reactivity. We evaluated whether a human cat chimeric fusion protein (GFD) composed of part of the human Ig G1 Fc fused to the major cat allergen (Fel d1) would function as allergen immunotherapy while not inducing acute allergic reactivity in mice sensitized to Fel d1. Injection of GFD 6 h prior to Fel d1 challenge acutely blocked systemic and skin reactivity to Fel d1 challenge while mice given subcutaneous immunotherapy with GFD at days 37, 38, and 39 showed inhibition of systemic, lung, and cutaneous reactivity to Fel d1 2 weeks later. GFD immunotherapy did not induce systemic reactivity. Overall, the Fcgamma-Fel d1 chimeric fusion protein blocked Fel d1-induced IgE-mediated reactivity but did not induce in vivo mediator release on its own; suggesting that this approach using allergen combined with Fc gamma1 so as to achieve inhibitory signaling may provide an enhanced form of allergen immunotherapy.     

A mutant of the major melon allergen, Cuc m 2, with reduced IgE binding capacity is a good candidate for specific immunotherapy

Hypoallergenic mutants with reduced IgE-binding capacity but which show a similar T-cell response to the corresponding natural allergen are ideal tools for immunotherapy, for preventing a possible anaphylactic shock. An IgE conformational epitope has been identified in Cuc m 2, the major allergen and profilin from melon. Since this epitope is highly conserved in most pollen profilins, it may contribute to an explanation of cross-reactivity between pollen and food profilins. Mutants (Mut 1 and Mut 2) were generated by changing specific residues of the Cuc m 2 epitope to alanine, produced in Escherichia coli, and purified by chromatographic methods. Mut 1 showed a slight reduction in IgE binding but an allergenic activity that was similar to recombinant Cuc m 2, as measured by basophil activation test (BAT) and skin prick test (SPT). By contrast, Mut 2 displayed a substantial reduction in IgE-binding capacity (57%) and positive responses, as determined by BAT (33%) and SPT (50%), when compared to those of rCuc m 2. However, the T-cell proliferation and cytokine production induced by Mut 2 and rCuc m 2 were similar. Thus, this mutant represent potential candidate for immunotherapy of profilin allergies.