High correlation of specific IgE sensitization between birch pollen, soy and apple allergens indicates pollen-food allergy syndrome among birch pollen allergic patients in northern China.

BACKGROUND: Birch pollen sensitization and associated pollen-food syndrome among Chinese allergic patients have not been investigated. METHODS: Sera from 203 allergic patients from the northern part of China and collected during February to July 2014 were investigated. Specific immunoglobulin E (IgE) against birch pollen extract Bet v and major birch pollen allergen Bet v 1 were measured using the ADVIA Centaur. The presence of major apple allergen Mal d 1 and soy bean allergen Gly m 4 specific IgE was measured by ImmunoCAP 100. RESULTS: Among the 203 sera, 34 sera (16.7%) had specific IgE to Bet v and of these, 28 sera (82.4%) contained Bet v 1-specific IgE. Among the 28 sera with Bet v 1-specific IgE, 27 sera (96.4%) contained Mal d 1-specific IgE and 22 sera (78.6%) contained Gly m 4-specific IgE. Of the 34 Bet v-positive sera, 6 sera (17.6%) contained no specific IgE for Bet v 1, Mal d 1, or Gly m 4. Almost all Bet v-positive sera were donated during the birch pollen season. CONCLUSIONS: The prevalence of birch allergy among patients visiting health care during pollen season can be as high as 16.7% in Tangshan City. The majority of Chinese birch allergic patients are IgE-sensitized to the major birch pollen allergen Bet v 1 as well as to the major apple allergen Mal d 1 and soy bean allergen Gly m 4. A relatively high number of patients (17.6%) are IgE-sensitized to birch pollen allergen(s) other than Bet v 1. The high prevalence of specific IgE to Mal d 1 and Gly m 4 among Bet v 1-sensitized patients indicates that pollen-food allergy syndrome could be of clinical relevance in China.

The Functional IgE-Blocking Factor Induced by Allergen-Specific Immunotherapy Correlates with IgG4 Antibodies and a Decrease of Symptoms in House Dust Mite-Allergic Children.

BACKGROUND: At present, there are no validated biomarkers reflecting or predicting the clinical efficacy of allergen-specific immunotherapy (AIT) . We aimed to investigate the correlations between clinical and immunological responses of patients undergoing house dust mite (HDM) AIT. METHODS: Sixty-nine children diagnosed with HDM allergic rhinitis and/or asthma received standardized Dermatophagoides pteronyssinus (Dp) subcutaneous AIT for 12 months. Twenty HDM-allergic children served as an open control group. Clinical symptom and medication scores were recorded and Dp-specific IgE, IgG4 and IgE-blocking factor were measured before AIT and after 4 and 12 months of AIT. RESULTS: Symptom scores decreased after 4 months and continued to decrease during 12 months of AIT. No differences in medication scores were observed between AIT and the control group during the study period. Levels of Dp IgG4 increased after 4 months and correlated to symptom scores at 12 months (r = -0.296, p = 0.013) of AIT. The Dp IgE-blocking factor increased after 4 months of AIT, and correlated with symptom scores at 4 months (r = -0.307, p = 0.010) and 12 months (r = -0.288, p = 0.016) of AIT. A strong correlation between Dp IgE-blocking factor and Dp IgG4 during AIT (4 months: r = 0.680; 12 months: r = 0.636, both p < 0.0001) was observed. Patients with IgE-blocking factor ≥0.2 after 4 months of AIT showed lower symptom scores at 12 months of AIT (p = 0.0093). CONCLUSIONS: Subcutaneous HDM AIT results in a decrease of allergic symptoms among HDM-allergic children. IgE-blocking activity increased after 4 months of AIT and correlated with clinical symptoms. A high IgE-blocking factor at an early stage of AIT is associated with fewer symptoms at a later stage of AIT.

Prevalence of sensitization to weed pollens of Humulus scandens, Artemisia vulgaris, and Ambrosia artemisiifolia in northern China.

OBJECTIVE: Weed pollens are common sources of allergens worldwide. The prevalence of weed pollen sensitization is not yet fully known in China. The purpose of this study was to investigate the prevalence of sensitization to weed allergens from Artemisia, Ambrosia, and Humulus in northern China. METHODS: A total of 1144 subjects (aged from 5 to 68 years) visiting our clinic from June to October 2011 underwent intradermal testing using a panel of 25 allergen sources. Subjects with positive skin responses to any pollen were further tested for their serum concentrations of IgE antibodies against Artemisia vulgaris, Ambrosia artemisiifolia, and Humulus scandens, and against the purified allergens, Art v 1 and Amb a 1. RESULTS: Of 1144 subjects, 170 had positive intradermal reactions to pollen and 144 donated serum for IgE testing. The prevalence of positive intradermal responses to pollens of Artemisia sieversiana, Artemisia annua, A. artemisiifolia, and H. scandens was 11.0%, 10.2%, 3.7%, and 6.6%, respectively. Among the intradermal positive subjects, the prevalence of specific IgE antigens to A. vulgaris was 58.3%, to A. artemisiifolia 14.7%, and to H. scandens 41.0%. The prevalence of specific IgE antigens to the allergen Art v 1 was 46.9%, and to Amb a 1 was 11.2%. The correlation between the presence of IgE antibodies specific to A. vulgaris and to the Art v 1 antigen was very high. Subjects with A. artemisiifolia specific IgE also had A. vulgaris specific IgE, but with relatively high levels of A. vulgaris IgE antibodies. There were no correlations between the presence of IgE antibodies to H. scandens and A. vulgaris or to H. scandens and A. artemisiifolia. CONCLUSIONS: The intradermal prevalence of weed pollen sensitization among allergic subjects in northern China is about 13.5%. Correlations of specific IgE antibodies suggest that pollen allergens from Artemisia and Humulus are independent sources for primary sensitization.

Specific IgG4 production during house dust mite immunotherapy among age, gender and allergic disease populations.

BACKGROUND: Specific IgG4 induced by allergen-specific immunotherapy (SIT) is an immunological marker related to the appearance of clinical tolerance. But specific IgG4 levels in different age, gender and allergic disease populations have not been fully investigated. METHODS: This study involved 226 children and 109 adults with allergic rhinitis and/or asthma receiving a 156-week course of Dermatophagoides pteronyssinus (Der p) subcutaneous SIT. Symptom and medication scores, forced expiratory volume after 1 s (FEV(1)) and Der p-specific IgG4 levels at weeks 0, 5, 10, 25, 52, 104 and 156 were analyzed. RESULTS: Rhinitis symptom and medication scores and FEV(1) % predicted in children showed significantly greater improvement than in adults at week 104 and 156 (p < 0.05). Levels of Der p-specific IgG4 showed a significant increase after 10 weeks of subcutaneous SIT (p < 0.0001) and continued to increase during the 156-week SIT period. Before SIT, the initial Der p-specific IgG4 level was higher in children than adults (p = 0.0004). The increase ratio of Der p-specific IgG4 was higher in children than adults at 52 weeks (p < 0.001) and 104 weeks (p = 0.0156) of SIT, and was higher in rhinitis compared to asthma patients at 156 weeks of SIT (p = 0.0244). There was no difference between males and females at any time points. CONCLUSION: Children are more responsive to SIT, demonstrating clinical and FEV(1) improvement and producing higher levels of allergen-specific IgG4 during a shorter SIT period compared to adults. Rhinitis patients show a higher increase in specific IgG4 compared to patients with asthma symptoms. The increase of Der p-specific IgG4 reflects a specific response of the immune system towards the SIT vaccine being administrated.

Indoor mite allergen levels, specific IgE prevalence and IgE cross-inhibition pattern among asthmatic children in Haikou, southern China.

BACKGROUND: Haikou locates in tropical island with unique mite propagation. The aim of this stuy is to determine mite allergens levels in Haikou, and to investigate the prevalence of mite specific IgE-sensitization and IgE cross-reactivity between house dust mites. METHODS: Allergen and antigen concentrations against six mite species were tested by enzyme-linked immunosorbent assay (ELISA). Specific IgE concentrations and cross-inhibitions were measured with ADVIA Centaur(®). RESULTS: Allergen or antigen Dermatophagoides pteronyssinus (Der p 1), Blomia tropicalis (Blo t) and Tyrophagus putrescentia (Tyr p) were detected in dust samples. Dermatophagoides farinae (Der f 1), Lepidoglyphus destructor (Lep d 2), and Acarus siro (Aca s) were found in very few samples. Specific IgE tests showed high prevalence of sensitizations against all tested mites with high IgE levels to Der p, Der f, and Blo t. Storage mites, Blo t, Tyr p, Lep d, and Aca s, could inhibit Der p from 0 to 50%. Storage mites could inhibit Der f between 30% and 100%. Der p IgE could be inhibited by Der f with up to 90%, and vice versa. Der p could inhibit Blo t from 40% to 80%. Blo t was able to fully inhibit IgE binding to Lep d, Tyr p, and Aca s compared to partial inhibition by Der p. CONCLUSIONS: Der p is the dominating mite and has the highest specific IgE prevalence among asthmatic children. Blo t represents an important source of storage mite sensitization and some patients may be independently sensitized to both Der p and Blo t. High prevalence of sensitization to Der f may be due to IgE-mediated cross-reactivity with Der p and Blo t.

Factors associated with allergen sensitizations in patients with asthma and/or rhinitis in China.

BACKGROUND: Allergen sensitization is influenced by genetic and environmental factors; however, the factors related to sensitizations in patients with rhinitis and asthma in China are largely unknown. This study investigated the factors associated with allergen sensitizations in patients with asthma and rhinitis in China. METHODS: A cross-sectional survey was performed in 6304 patients with asthma and/or rhinitis from four regions of China. Patients completed a standardized questionnaire related to respiratory and allergic symptoms, family history of allergic diseases, smoking history, environmental exposure, and eating behaviors. They underwent skin-prick tests (SPTs) with 13 common aeroallergens. Blood samples were collected from 2268 of patients for specific IgE (sIgE) measurements against 16 common aeroallergens. RESULTS: Patients with both asthma and rhinitis had higher prevalence of SPT and sIgE positivity to most allergens than those with asthma or rhinitis alone (p < 0.0001). Male gender, family history of allergic rhinitis, air-conditioner usage, sleeping on a mattress, and frequently eating meat were associated with increased risk of SPT and sIgE positivity. Using air-conditioner and sleeping on a mattress were further found to be associated with sIgE positivity to mites and molds. However, increased age and fish, fruit, and raw vegetable intake decreased the risk of SPT and sIgE positivity. CONCLUSION: Family history of allergic rhinitis, male gender, using an air conditioner, sleeping on a mattress, and frequent meat consumption are risk factors for allergen sensitizations, whereas increased age and frequent fish, fruit, and raw vegetable consumption may protect patients with asthma and/or rhinitis from developing sensitizations in China.

House dust mite and storage mite IgE reactivity in allergic patients from Guangzhou, China.

BACKGROUND: In China, house dust mites are important inducers of allergic disease. The importance of allergens from storage mites is less well known. OBJECTIVE: The aim of this study is to access the prevalence of house dust mite and storage mite sensitization and investigate the IgE crossreactivity between house dust mite and storage mite. METHOD: The skin prick test (SPT) and specific IgE against the mite species D. pteronyssinus, D. farinae, B. tropicalis, Lepidoglyphus destructor, Glycyphagus domesticus, Tyrophagus putrescentiae and Acarus siro were measured. Included were 412 patients with asthma and/or rhinitis for SPT, 244 for specific IgE and 29 sera for IgE inhibition studies. RESULTS: The positive SPT prevalence for D. pteronyssinus was 80.3% and for D. farinae 83.7%. The specific IgE prevalence for D. pteronyssinus was 61.1% and for D. farinae 60.2%. The storage mite species, B. tropicalis and T. putrescentiae had the highest positive SPT prevalence, 66% and 63%, respectively. The specific IgE prevalence was highest for B. tropicalis and G. domesticus, 41% and 37%, respectively. Both SPT and specific IgE levels were much higher for house dust mites compared to storage mites. Inhibition measurements showed that none of the storage mites could fully inhibit the specific IgE against D. pteronyssinus. Only in half of the sera could D. pteronyssinus fully inhibit the IgE against L. destructor and G. domesticus while inhibition of the other storage mites were much lower. Nearly all the specific IgE against storage mites could be inhibited by the other storage mites, though B. tropicalis showed a slightly different pattern from the other storage mites. CONCLUSION: IgE reactivity against storage mites in Chinese patients is due to both storage mite specific IgE and due to IgE mediated crossreactivity to D. pteronyssinus.

Influence of degree of specific allergic sensitivity on severity of rhinitis and asthma in Chinese allergic patients.

BACKGROUND: The association between sensitizations and severity of allergic diseases is controversial. OBJECTIVE: This study was to investigate the association between severity of asthma and rhinitis and degree of specific allergic sensitization in allergic patients in China. METHOD: A cross-sectional survey was performed in 6304 patients with asthma and/or rhinitis from 4 regions of China. Patients completed a standardized questionnaire documenting their respiratory and allergic symptoms, their impact on sleep, daily activities, school and work. They also underwent skin prick tests with 13 common aeroallergens. Among the recruited subjects, 2268 provided blood samples for serum measurement of specific IgE (sIgE) against 16 common aeroallergens. RESULTS: Significantly higher percentage of patients with moderate-severe intermittent rhinitis were sensitized to outdoor allergens while percentage of patients sensitized to indoor allergens was increased with increasing severity of asthma. Moderate-severe intermittent rhinitis was associated with the skin wheal size and the level of sIgE to Artemisia vulgaris and Ambrosia artemisifolia (p < 0.001). Moderate-severe asthma was associated with increasing wheal size and sIgE response to Dermatophagoides (D.) pteronyssinus and D. farinae (p < 0.001). Moderate-severe rhinitis and asthma were also associated with increase in number of positive skin prick test and sIgE. CONCLUSIONS: Artemisia vulgaris and Ambrosia artemisifolia sensitizations are associated with the severity of intermittent rhinitis and D. pteronyssinus and D. farinae sensitizations are associated with increasing severity of asthma in China. Increase in number of allergens the patients are sensitized to may also increase the severity of rhinitis and asthma.

Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1.

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.

Allergen micro-array detection of specific IgE-reactivity in Chinese allergy patients.

BACKGROUND: Allergen micro-arrays are powerful tools for screening of serum IgE-reactivity. In this study allergen micro-arrays were used to identify dominating IgE-binding allergens and cross-reactivity patterns among selected Chinese allergy patients. METHODS: The study was conducted using patient sera from the cities of Guangzhou, Nanjing, Chengdu and Shenyang. In total 100 sera with Dermatophagoides pteronyssinus (Der p) specific IgE-levels higher than 50 kU/L were selected for testing against 103 individual allergens. RESULTS: Among 100 selected patients, 95% showed IgE-reactivity towards house-dust mite allergens Dermatophagoides farinae (Der f) 1, Der f 2 and Der p 2 and 94% were IgE positive against Der p 1, and 60% of sera contained IgE reacting against allergen Euroglyphus maynei (Eur m) 2. IgE against cat allergen, Felisdomesticus (Fel d) 1, was seen in 20%. Only 2% showed specific IgE-reactivity to Der p 10, a panallergen belonging to the tropomyosin family. Serum IgE-reactivity towards other allergens was in general low. IgE-reactivity against pollen allergens showed geographic differences. CONCLUSIONS: This study clearly confirms that group 1 and group 2 are major allergens of house dust mites. These selected house-dust mite allergy patients are close to being mono-sensitized. Der p 10 is not an important allergen for cross-reactivity. Specific IgE-sensitization towards pollen allergens is low in southern China compared to other regions. The prevalence of food and stinging insect allergens known to give rise to IgE-mediated cross-reactivity is 2% or less.

The specific IgE reactivity pattern of weed pollen-induced allergic rhinitis patients.

CONCLUSION: Specific immunoglobulin E (IgE) reactivity towards the major mugwort allergen Art v 1 is a good indicator for Art v sensitization. Allergens from the ragweed species Amb t and Amb a possibly share common IgE-binding epitopes. OBJECTIVE: The aim of this study was to investigate the reactivity pattern of IgE in Chinese patients with weed pollen-induced allergic rhinitis. METHODS: Sera from 50 weed pollen-induced allergic rhinitis patients were tested for specific serum IgE reactivity against allergenic extracts of mugwort (Artemisia vulgaris, Art v), short ragweed (Ambrosia artemisiifolia, Amb a), giant ragweed (Ambrosia trifida, Amb t), and single allergens of Art v 1, Art v 3, Amb a 1, and profilin. RESULTS: Sera from 88% of the patients demonstrated positive specific IgE reactivity to Art v, and of these 82% were positive to Art v 1. Sera from 38% of the patients showed positive specific IgE reactivity to both ragweed species Amb t and Amb a. A strong correlation was found between the specific IgE levels of Amb t and Amb a. Of the Amb a IgE-positive patients, 38% were positive for Amb a 1. Of all patient sera tested, 12% were specific IgE-positive to profilin.

[Sequence polymorphism of major house dust mite allergens].

OBJECTIVE: To determine the sequence variation of the groups 1 and 2 allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae in different geographical regions. METHODS: RNAs were isolated from the whole bodies, eggs and entire culture of mites (D. farinae) from South China and European commercialized D. pteronyssinus and D. farinae. Sequences of prepeptide, propeptide and mature peptide fragments in group 1, and prepeptide and mature peptide fragments in group 2 were determined from cDNA produced by PCR. All the sequences were compared with the corresponding standards in GenBank. RESULTS: Der p 1.0105 was the major variant found in European commercialized Der p 1 clones, where amino acid variations appeared sporadic, except for frequent substitutions shown in residues 124, 50 and 19, and eight novel homologous variants were identified. Derp 2.0104 was mostly detected in clones from European commercialized Der p 2, where amino acid substitutions were predominant in residues 40, 47, 111 and 114, and six novel homologous variants were identified. Very few amino acid sequence substitutions were found among South China based Der f 1 clones, where three novel homologous variants were identified. As compared with European commercialized ones, a number of homologous variants were recognized unique to South China based Der f 2 clones where six novel homologous variants were identified. There was no significant difference in major allergens among variants from the whole mite bodies, eggs or the entire culture. CONCLUSION: There is a difference among the predominant variants of major house dust mite allergens in different geographical regions.

Allergens as eukaryotic proteins lacking bacterial homologues.

Only a small number of protein homologues cause the majority of allergies. There is no consensus structure or other obvious common denominator discriminating the few proteins that are allergens from thousands of other, non-allergenic proteins. By database sequence homology searching, we here show that to date known allergen sequences have no or few bacterial homologues, in contrast to randomly selected control protein sequences. This finding suggests a novel common denominator for allergens of potential use for allergen prediction programs. A possible interpretation of this finding is that allergens are proteins which are exposed to the immune system and which lack bacterial homologues. This interpretation is discussed in relation to the many observations that allergies coincide with a delayed establishment of infant gut flora.

Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom.

Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved cavity near the active site is involved in binding of the substrate. Surface epitopes and putative glycosylation sites have been compared with those of two other major group 2 allergens from Apis mellifera (honeybee) and Dolichovespula maculata (white-faced hornet). The analysis suggests that the harboured allergic IgE-mediated cross-reactivity between Ves v 2 and the allergen from D. maculata is much higher than that between Ves v 2 and the allergen from A. mellifera.

Standardization of allergen-specific immunotherapy vaccines.

In the 1970s and 1980s, scientific methods were introduced in the standardization of allergen vaccines and, in combination with improved documentation of the clinical benefits obtained using standardized vaccines, specific allergy treatment as a scientifically based, reproducible, and safe treatment for allergic disease was established. This article describes important issues in the control of source materials and vaccine preparation as part of the European standardization of allergen vaccines, and also includes a discussion of vaccines that are based on recombinant allergens, which may appear on the market in the near future.

Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes.

Novel approaches for the prevention of allergy are required, because of the inevitably increasing prevalence of allergic diseases during the last 30 years. Here, a recombinant chimeric protein, which comprises the whole amino acid sequences of three bee venom major allergens has been engineered and used in prevention of bee venom sensitization in mice. Phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 3) fragments with overlapping amino acids were assembled in a different order in the Api m (1/2/3) chimeric protein, which preserved entire T cell epitopes, whereas B cell epitopes of all three allergens were abrogated. Accordingly, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced in humans. Supporting these findings, the Api m (1/2/3) induced 100 to 1000 times less type-1 skin test reactivity in allergic patients. Treatment of mice with Api m (1/2/3) led to a significant reduction of specific IgE development towards native allergen, representing a protective vaccine effect in vivo. These results demonstrate a novel prototype of a preventive allergy vaccine, which preserves the entire T cell epitope repertoire, but bypasses induction of IgE against native allergen, and side effects related to mast cell/basophil IgE FcepsilonRI cross-linking in sensitized individuals.

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen.

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.

Molecular characterization of major cat allergen Fel d 1: expression of heterodimer by use of a baculovirus expression system.

Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization.