IgE Antibody Responses to Recombinant Allergens of Blomia tropicalis and Dermatophagoides pteronyssinus in a Tropical Environment

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1 Research Trends 233 IgE Antibody Responses to Recombinant Allergens of Blomia tropicalis and Dermatophagoides pteronyssinus in a Tropical Environment by Silvia Jiménez, Leonardo Puerta, Dary Mendoza, Kaw Yan Chua, Dilia Mercado, & Luis Caraballo Background: Dermatophagoides pteronyssinus and Blomia tropicalis are the major sensitizer mite species in tropical regions. Several allergens from these species have been obtained by molecular cloning. In order to use them in clinical practice, evaluation of their allergenic potential in different populations is mandatory. This study evaluates the clinical significance and allergenicity of several recombinant allergens from B. tropicalis and D. pteronyssinus in asthmatic patients from a tropical environment. Methods/Data base: Specific IgE to allergenic extracts of D. pteronyssinus and B. tropicalis and to the recombinant allergens BtM, a peptide homologue to Blo t 5, Blo t 12, Blo t 13, Der p 2, Der p 5, Der p 7, and Der p 1 as well as the natural Der p 1 (nder p 1), was determined by radioallergosorbent test (RAST) in sera from 9 asthmatic patients and 1 healthy controls. In addition, SPT was performed in a selected group of patients. Results: The prevalence of reactivity to D. pteronyssinus and B. tropicalis extracts was 86.6% and 84.4%, respectively. The frequency of positive RAST to each D. pteronyssinus allergen was as follows: Der p 2, 68.9%; Der p 1, 64.4%; Der p 5, 37.8%; Der p 1, 16.7% and Der p 7, 15.6%. The frequency of positive RAST to B. tropicalis allergens was as follows: BtM, 42.2%; Blo t 12, 16.7% and Blo t 13, 11.1%. Together, Der p 1, Der p 2, and Der p 1 were able to detect 93.5% of 27 Hogrefe & Huber Publishers DOI: 1.127/ D. pteronyssinus allergic subjects. Via skin testing, all 8 mite allergens induced positive reactions in the selected allergic patients and negative results among the nine non-allergic subjects. Conclusions: The recombinant allergens tested are potentially useful as tools for in vitro and in vivo testing of mite allergy. Together, three recombinant allergens were able to detect 93% of D. pteronyssinus allergic subjects. Keywords: recombinant allergens, mite allergy, B. tropicalis, D. pteronyssinus Allergy Clin Immunol Int J World Allergy Org 27; 19: Introduction Blomia tropicalis and Dermatophagoides pteronyssinus are the most prevalent domestic mites in house dust of the tropical and subtropical regions [1, 2], where allergic sensitization to these mites is high. In these regions some patients become sen-

2 234 Research Trends sitized to species-specific allergens [3-6], suggesting that molecules from both mites increase the allergenic potential of house dust and should be included as reagents for the diagnosis and treatment of allergic respiratory diseases. 7 6 More than 2 allergens have been detected in the extract of B. tropicalis and D. pteronyssinus; most of them have been obtained and characterized by molecular cloning providing important reagents for molecular and epidemiological studies [7]. The progress in obtaining recombinant allergens with biological properties similar to the natural equivalent has stimulated their clinical use [8, 9]. An admixture of recombinant allergens, representing most of the allergenic activity of the natural extract, could eventually replace the natural preparations for diagnostic purposes. In addition, the use of recombinants for tailor-made immunotherapy, based on specific sensitization to allergenic molecules, has been proposed. Frequency (%) Der p 2 Der p 1 Der p 5 Der p 1 Der p 7 Allergens BtM Blo t 12 Blo t 13 This study investigates the clinical significance of several recombinant allergens from B. tropicalis and D. pteronyssinus in 9 asthmatic patients from a tropical environment. We evaluate their potential use in the diagnosis of allergic respiratory diseases in the tropics. Methods Subjects A total of 19 non-immunotherapy-treated asthmatic patients (mean age 35.7 years, range 8-62) living in the urban area of Cartagena, Colombia were selected, having a radioallergosorbent test (RAST) and skin prick test (SPT) positive to standardized B. tropicalis and D. pteronyssinus body extracts. Asthma was defined according to the Global Initiative for Asthma Criteria (GINA) [1]. Nine negative control sera were obtained from healthy subjects (mean age 29.7 years, range 12-58), with no history of atopy and negative RAST and SPT to the mite allergenic extracts. The investigation was approved by the Bioethics Committee of the Medical School of the University of Cartagena. Informed consent was obtained from all patient and healthy subjects. B. tropicalis and D. pteronyssinus Allergenic Extracts The B. tropicalis extract was prepared from whole body mite as described previously [11]. The D. pteronyssinus extract was obtained from Greer Laboratories (Lenoir, NC, USA), at 1, AU/ml. Purified Allergens Blo t 13 and BtM, a recombinant molecule of 73 amino acids that shows 78.1% identity with the carboxi terminal end of Blo t 5, were obtained as GST-fusion protein from E. coli/ Figure 1. Frequency of IgE reactivity to allergens from B. tropicalis and D. pteronyssinus in asthmatic patients as detected by RAST. pgex-4t-3-induced culture [12, 13]. Blo t 12 was expressed in the yeast Pichia pastoris using ppic 9 vector [14]. The recombinant allergens Der p 2, Der p 5, Der p 7, and Der p 1 were produced as described previously [15-17]. Natural Der p 1 (nder p 1) was obtained highly purified from natural mite culture. For control purposes, recombinant GST was obtained from IPTG-induced culture of E. coli transformed with pgex- 2T vector, without any cdna mite allergen insert. Determination of Allergen-Specific IgE Antibodies Specific IgE to purified allergens was determined in 9 sera from the selected allergic population by RAST as previously described [18]. Briefly, microtiter plates were coated with 25 ng of allergens or GST per well. 1μL of each serum sample was added to the wells, incubated overnight in a humid chamber, washed three times and incubated with 1μL of 125 I-labeled horse anti-human IgE (Kallestad Diagnosis Inc, Chaska, USA). After overnight incubation, the plates were washed three times, and each well was counted in a gammacounter (Gamma 55 Beckman). All assays were done in triplicate, and the results were expressed as the mean % of total count bound (TCB). The TCB equal or higher to.5% (mean + 2 SD of control sera) was considered positive. For determination of IgE antibodies to B. tropicalis and D. pteronyssinus, microtiter plates were coated with the allergenic extracts and processed as described above. Skin Prick Test For skin testing, B. tropicalis extract was used 5% glycerinated at.23 mg/ml, and D. pteronyssinus extract at 1, AU/mL. A 5% glycerinated and 2 mm filtered solution of each recombinant allergen was prepared with the following concentration: BtM, Blo t 12, Blo t 13, nder p 1, Der p 5, and GST were used at 25 ng/ml; Der p 2, Der p 7, and Der p 1 were

3 Research Trends 235 Table 1 SKIN PRICK TEST RESULTS TO B. tropicalis ALLERGENS B. tropicalis Patient extract BtM Blo t 12 Blo t 13 Figure 2. Correlation of specific IgE levels (% TCB) to Der p 1 and Der p 2 in sera from mite allergic patients. Mean TCB% (95% CI) HC neg ND neg HC ND neg HC ND neg HC neg ND neg HC ND neg HC neg neg HC ND neg HC ND 2 HC neg ND neg HC ND neg HC ND 4 HC neg 6 neg A14 9 neg neg 3 HC neg 15 HC neg neg HC neg neg HC neg HC neg neg neg HC neg 7 neg HC neg ND neg HC neg HC765 7 neg neg neg HC neg neg HC neg 3 neg HC neg neg neg HC neg neg neg * ND = not done BtM Blo t 12 Blo t 13 Der p 1 Der p 2 Allergen Der p 5 Der p 7 Der p 1 Figure 3. The strength of IgE response (mean of % TCB) to allergens as detected by RAST, among RAST-positive subjects. Results used as GST-fusion proteins at 75 ng/ml. A drop of allergen solution was pricked on the forearm using a sterile lancet. Histamine dihydrocloride and diluent were used as positive and negative controls, respectively. A wheal of 3 mm of diameter or greater, obtained 15 min after application of the allergen was considered positive. For SPT to B. tropicalis allergens 26 patients were selected, and 19 were selected for SPT to D. pteronyssinus allergens. The 9 nonallergic individuals were also skin prick tested. Statistical Analysis Statistical analysis and Spearman rank correlation coefficient of the RAST data were performed using SPSS 13 for Windows (SPSS Inc., Chicago, IL, USA). IgE Antibodies to D. pteronyssinus and B. tropicalisextracts A total of 76 sera (84.4%) were positive to B. tropicalis and 78 (86.6%) were positive to D. pteronyssinus; 75 sera (75%) had IgE to both D. pteronyssinus and B. tropicalis extracts. There was a statistically significant correlation between IgE levels to each mite species (p <.1). Eight sera react only to D. pteronyssinus and seven only to B. tropicalis. Five sera positive to both extracts were negative to all the recombinants tested. In the nonallergic control group, specific IgE antibodies to mite extracts or any of the recombinants were undetectable. IgE Antibodies to Purified Allergens The frequency of positive RAST to allergen from D. pteronyssinus was as follows: Der p 2, 68.9%; Der p 1, 64.4%;

4 236 Research Trends Table 2 Skin Prick Test results to D. pteronyssinus Allergens D. pteronyssinus Patient extract Der p 1 Der p 2 Der p 5 Der p 7 Der p 1 HC1958 neg neg neg neg neg neg HC neg 3 ND ND HC neg 7 neg neg neg HC A14 1 neg 1 neg neg neg HC neg neg neg neg HC neg 5 neg neg neg HC neg 3 HC neg neg ND ND HC neg 1 neg neg neg HC neg neg neg neg neg HC1133 neg neg neg neg neg neg HC neg neg neg neg HC HC neg neg 3 neg 5 HC neg neg 3 neg 4 HC neg neg neg neg neg HC neg neg neg neg neg HC neg neg neg neg neg * ND = not done 2 2 Mean wheal diameter (mm) Mean wheal diameter (mm) Blo t 13 Dp extract Der p 1 Der p 2 Der p 5 Der p 7 Der p 1 Bt extract BtM Blo t 12 Blo t 13 Figure 4. The strength of IgE response (mean of wheal diameter) to allergens in SPT, among SPT positive subjects. Der p 5, 37.8%; Der p 1, 16.7%; and Der p 7, 15.6%. Frequency of positive RAST to B. tropicalis allergens was as follows: BtM, 42.2%; Blo t 12, 16.7%; and Blo t 13, 11.1%, (Figure 1). Nine sera (1%) were positive to GST. Five out of ten positive sera to Blo t 13 exhibited specific IgE levels higher than 2% TCB. In contrast, IgE levels to Blo t 12 were usually low. A significant correlation between specific IgE levels to Der p 1 and Der p 2 (Spearman r =.61, p <.1) was also found (Figure 2). Of the 78 D. pteronyssinus allergic sera, 48 (61.5%) were positive to these recombinants. Der p 1 and Der p 2 account for most of the reactivity frequency (92.3%) among D. pteronyssinus-allergic patients, and taken together these two allergens plus Der p 1 detected 93.5% of the D. pteronyssinuspositive sera. In contrast, the combination of the three B. tropicalis allergens (BtM, Blo t 12, and Blo t 13) identified only 52.6% of B. tropicalis-positive sera. Some 38 sera were allergic to BtM, 29 of which (76.3%) were also IgE reactive to Der p 5; 34 sera were allergic to Der p 5, 29 thereof (85.%) were IgE reactive to BtM. There was also a significant correlation between IgE antibodies levels to BtM and Der p 5 (Spearman r =.64, p <.1). The mean of specific IgE levels to each allergen among RAST positive subjects is depicted in Figure 3.

5 Research Trends 237 Skin Prick Test Results of the SPT are shown in Tables 1 and 2. All recombinants allergens induced positive reactions in allergic patients. All nonallergic controls had negative SPT (data not shown). With D. pteronyssinus allergens, the frequency of positive SPT was lower than that obtained by RAST except for Der p 1, which showed 29.4% of positive SPT and 16.7% with RAST. With B. tropicalis allergens, the frequency of reactivity in SPT was higher than that obtained by RAST. Figure 4 shows the mean wheal diameters (mm) among positive SPT. In general, the intensity of the IgE response shows the same pattern in RAST and SPT. In addition, there was a significant difference between the means of all D. pteronyssinus allergens and all B. tropicalis allergens, but not between the means with mite extracts. There were no positive skin test reactions to GST. Discussion Our results concerning IgE reactivity to the allergenic extract of D. pteronyssinus and B. tropicalis support the idea that sensitization to both mite species is high in tropical areas. We also found that 8% to 9% of allergic patients were sensitized exclusively to B. tropicalis or D. pteronyssinus, which is similar to the values of 1% and 11% for exclusive sensitization to each one of these mite species in a study of Venezuelan patients [5], which underlines the importance of including allergens from both mite species for diagnostic purposes. Only 57% of our patients allergic to D. pteronyssinus were double positive to Der p 1 and Der p 2, which suggests that other allergens could play a role in inducing sensitization to this mite in the studied population. In fact, three patients were positive to Der p 1 only, and three patients were positive to Der p 5 only, although no unique IgE reactivity to Der p 7 was found. This result is similar to that obtained in asthmatic subjects in Uberlandia (Brazil) [19], where 6% of patients were double positive to Der p 1 and Der p 2. However, it has been reported foro Middle Europe that more than 95% of mite allergic patients were mainly sensitized to Der p 1 and Der p 2, and that diagnostic test containing these allergens plus the highly cross-reactive allergen Der p 1 may improve the diagnostic selection of patients for immunotherapy with D. pteronyssinus extracts [2]. It has been suggested that the majority of mite-allergic subjects elicit an IgE response to around five components of the allergenic extract [17, 21], and that some of them may be crossreactive. Therefore, it could be anticipated that an admixture of few allergens, including those species-specific and cross-reactive, could replace the crude allergenic extract for diagnostic and therapeutic purposes. In this regard, determining the profile of IgE response to the allergenic components of clinically relevant mite species in the tropics is an important step toward the goal of providing better immunotherapy. Our finding confirms that Der p 1 and Der p 2 are very important sensitizers in a tropical environment, and suggest that these two major allergens plus one minor allergen from the same mite could be used for diagnosing D. pteronyssinus allergy. Reactivity to Der p 1, the D. pteronyssinus tropomyosin, in our allergic population was 16.7%, which is higher than the 5.6% reported in [22], the 6% reported in a Middle European mite allergic population [2], and the 8.6% reported in hospitalized allergic children from Australia [23]. In addition, in this study we report frequency of reactivity and IgE levels to Der p1 slightly higher than those for Der p 7, which was classified as midrange allergen in the Australian study [23]. These differences could be influenced by the co-exposure to cross-reacting tropomyosin from helminths, cockroach, other species of house dust mites, etc. Therefore, the role of allergens such as Der p 1 should be further analyzed regarding the contribution of other factors, including the exposure to different allergen sources that might influence the immunologic responsiveness in allergic populations from tropical and subtropical regions. The frequency of positive RAST to GST in the allergic group (1%) is close to the 16% reported in patients from Venezuela [24]. The GST encoded by the pgex-2t vector is from Schistosoma, and since there is no schistosomiasis in our population, it is likely that this result also reflects exposure to homologous allergens from mites and other sources. Indeed, the GST from D. pteronyssinus (Der p 8) showed 75% and 65% IgE reactivity in allergic subjects from Malaysia and Singapore, respectively, and 47% of IgE cross-reactivity between this molecule and cockroach GST has been described [25]. The prevalence of IgE reactivity to the peptide BtM (42%) is lower than the rate obtained using the whole molecule Blo t 5 in Taiwanese (91.8%) and in Malaysian (73.5%) populations [26]. We also found a higher frequency of reactivity to Blo t 5 than to BtM (76.7% vs. 64.2%) in 43 mite-allergic individuals from Cuba [unpublished]. These results suggest that the amino-terminal portion of Blo t 5 contains important IgE epitopes absent in the BtM molecule. Even thought BtM is a partial clone, it elicited stronger IgE response than Der p 5, and the number of sera sensitized to BtM without sensitization to Der p 5 was greater than the sera sensitized to Der p 5 without sensitization to BtM, supporting the major role of B. tropicalis in the tropics, where the group 5 allergen from B. tropicalis is a more important inducer of IgE response than the group 5 allergen from D. pteronyssinus. The strength of allergic response to Blo t 13 was high in 5 of 1 patients sensitized to this allergen, similar to the results of the major allergens Der p 1 and Der p 2. This finding is in agreement with our previous work, where we found that, in a particular serum, Blo t 13 was able to inhibit the IgE reactivity to B. tropicalis extract in 62% [13]. In addition, it supports the notion that admixtures of recombinant allergens to be used to replace the allergenic extracts in diagnosis should contain both minor and major allergens. We found different frequencies of positive test with RAST and SPT. With B. tropicalis allergens, positive SPTs were more frequent, while positive RASTs were more frequent with most

6 238 Research Trends of D. pteronyssinus allergens. Although SPT is considered more sensitive than in vitro testing for specific IgE, as was the case of B. tropicalis allergens, the small number of patients with SPT makes it difficult to conclude accurately which test is more sensitive. For SPT we used the same concentration of recombinant allergens used in studies with population from other regions [26, 27]; nevertheless, further studies, including testing a larger number of patients, are needed to clarify whether standardization of the recombinant allergens for SPT by endpoint titration in a tropical population could change the difference in the frequency of reactivity between RAST and SPT. No adverse reactions were observed in the allergic or control subjects who were skin tested. Therefore, we can conclude that recombinant allergens from B. tropicalis and D. pteronyssinus and the natural Der p 1 are useful for in vitro and in vivo diagnostic tests of mite allergy diseases. 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Clin Exp Allergy 22; 32: Dr. Leonardo Puerta (to whom correspondence should be addressed) is with the Instituto de Investigaciones Inmunológicas, Universidad de Cartagena, Campus de Zaragocilla, Edificio Biblioteca, Piso 1, Cartagena, Colombia (fax , lpuertall@yahoo.com). Silvia Jiménez, Dary Mendoza, Dilia Mercado, and Luis Caraballo are with the Instituto de Investigaciones Inmunológicas, Universidad de Cartagena, Colombia. Kaw Yan Chua is with the School of Medicine, Department of Pediatrics, National University of Singapore, Singapore.