Characterization of a major house dust mite allergen Der p 22 among Malaysian adult population

Abstract

Background

House dust mites (HDM) are a major trigger factor of allergic diseases worldwide.

Objective

To characterize the group 22 allergen from the HDM Dermatophagoides pteronyssinus among Malaysian atopic adults.

Methods

Recombinant Der p 22 and 33 alanine mutants were expressed in Escherichia coli and purified. Specific IgE levels to group 22 allergens were measured in serum samples from a combined cohort of Malaysian adults (n = 176) and participants from the SMCGES sub-cohort (n = 755), with responses classified by ImmunoCAP classes. Age- and gender-adjusted multivariate logistic regression was used to assess associations between Class 3+ group 22-specific IgE responses (>3.5 IU/mL) and clinical diagnoses of asthma, allergic rhinitis (AR), and atopic dermatitis (AD). IgE inhibition assays were performed to evaluate the presence of unique epitopes in Der p 22 relative to its homologue Der p 2. Epitope mapping was performed using 33 single-alanine substitution mutants of Der p 22. Statistical analyses were carried out using GraphPad Prism and logistic regression models.

Results

Recombinant Der p 22 was expressed and purified as a 17.2 kDa protein. Among 149 atopic Malaysian adults, 54% had detectable sIgE to Der p 22. IgE inhibition assays demonstrated partial cross-reactivity between Der p 22 and Der p 2. High-level group 22-specific IgE responses (Class 3+) were associated with an increased risk of asthma (OR = 1.73, 95% CI = 0.98–3.00, p = 0.0524) and significantly associated with the presence of atopic comorbidities including AR and AD (all p < 0.05). Epitope mapping identified to 6 critical residues, K10, H35, R43, D106, R117 and R130, where alanine substitution led to a marked reduction in IgE binding (median reduction >95%).

Conclusion

Der p 22 is a clinically relevant HDM allergen in the Malaysian adult population. It harbours both unique and shared IgE epitopes with Der p 2, highlighting its value for inclusion in molecular diagnostic panels to improve allergen sensitization profiling. Alanine mutation to 6 amino acid residues of Der p 22 significantly reduced IgE binding, suggesting its potential as a candidate hypoallergen molecule in allergen-specific immunotherapy.

Introduction

At least 20% of the world's population is affected by allergies.¹ Dust mites from the Pyroglyphidae family, Dermatophagoides pteronyssinus and D. farinae are the most significant allergy-causing mites worldwide.^2,3 Among the 34 allergen groups identified so far from D. pteronyssinus (www.allergen.org), several allergens, namely group 1, group 2 and group 23, have been identified as major allergens.4, 5, 6 Collectively, they react to the IgE antibodies of 50–70% of all dust mite-allergic individuals.⁷ The characterization of new dust mite allergens will provide a more detailed understanding of their contribution in terms of sensitization and clinical implications among atopic individuals. The accurate knowledge of an individual's unique allergen sensitization profile would pave the way to improved and more accurate allergy therapeutics in the future.

The IgE-reactivity profiles of a group 22 allergen from D. farinae, Der f 22 were characterized in 2018.⁸ Der f 22 was reported to have specific IgE (sIgE) reactivity of 42% (in 106 of 253 patients) among the allergic individuals tested. Der f 22 was identified as a paralogue of Der f 2, the major allergen of D. farinae, based on the high three-dimensional structural similarity between both allergens.⁸ Interestingly, Der f 22 harbors clinically important and unique epitopes compared to Der f 2, suggesting the importance of characterizing new allergens.⁸

Recently, a research team from China reported on the preliminary characterization of Der p 22 among a paediatric asthmatic population.⁹ In that study, Der p 22 was identified as a major allergen, with specific IgE reactivity observed in 93.75% of tested individuals. Further in vivo and in vitro experimentation confirmed that Der p 22 elicited an allergy-promoting Th2 milieu, and induced apoptosis in culture lung cells.⁹

In this study, we aim to further characterize the specific IgE reactivity of Der p 22 in an adult atopic population. Additionally, Der p 22 will be assessed for the presence of unique and cross-reactive IgE epitopes in comparison to its structural homologue, Der p 2, and fine mapping of its IgE epitopes will be carried out. Correlations between Der p 22 sIgE and the total IgE titer and sensitization patterns of atopic individuals will also be investigated.

Methods

Study participants and ethical approval

This study comprises 2 ongoing and non-overlapping cohorts: the Malaysian cross-sectional young-adult cohort (n = 176) and the Singapore/Malaysia Cross-sequential Genetics and Epidemiological Study (SMCGES) cohort (n = 755). Participants in the Malaysian young adult cohort were recruited from Sunway University (SU), Malaysia, in November 2019. In contrast, participants in the SMCGES cohort were recruited from 3 universities: the University of Tunku Abdul Rahman (UTAR), Malaysia (February 2016 to October 2018); SU (from November 2019, ongoing); and the National University of Singapore (NUS) (from August 2005, ongoing). For both cohorts, all consenting participants filled in a validated questionnaire with questions on their demographics, medical history, environmental allergen exposure, and anthropometric measurements. This questionnaire was designed based on the guidelines of the Allergic Rhinitis Impact on Asthma (ARIA)^10,11 and the International Study of Asthma and Allergies in Childhood (ISAAC).¹² Ethical approval for this study was obtained from SU (Reference: SUREC2019/029), UTAR (Reference: U/SERC/03/2016), and NUS (Reference: 07–023, 09–256, 10–445, 13–075, B-10-343, and H-18-036). Recruitment procedures were conducted in accordance with the Helsinki Declaration, and written informed consent was obtained from all participants.

Skin prick test (SPT)

The patients gave their written consent before undergoing skin prick tests (SPT). Filter sterilized 0.02 mg/mL of allergen in buffer containing 50% glycerol was used to carry out SPT. SPT was conducted using 6 allergen extracts, Dermatophagoides pteronyssinus, Blomia tropicalis, Periplaneta americana, Blatella germanica, Elaeis guineensis and Curvularia spp. The size of the wheal and erythema was measured after 15 min. Histamine (10 mg/mL, Unison) was used as the positive control. Positive SPT results were defined as a wheal diameter of 3 mm or more compared to the negative control (saline).

Cloning, expression and purification of group 2 and 22 dust mite allergens

Mature genes coding for group 22 and group 2 from 2 dust mite species (D. pteronyssinus and D. farinae) were cloned into the pET 28b expression vector containing a hexa-histidine-tag by GenScript, UK. The mature proteins (Genebank accession number A0A291KZA0) were overexpressed as recombinant proteins using the E. coli expression system and purified as described previously.⁸ The Der p 22 allergen has been registered in the IUIS database as Der p 22.0101 (https://www.allergen.org/viewallergen.php?aid=1164).

Generation of rDer p 22 mutants

The 3D modelled structure of Der p 22 (Accession number: A0A291KZA0) was obtained using the mature protein sequence with ExPASy homology-modelling sever SWISS MODEL using the solved X-ray structure of a related allergen, Der f 2 (PDB: 1XWV.1.A) as a template. Based on previous literature demonstrating that the majority of dust mite allergen IgE epitopes were polar and charged surface exposed residues,^4,8,13, 14, 15, 16, 17 we identified 33 amino acid residues on Der p 22 that fit these criteria. Plasmid constructs of Der p 22 with single alanine mutations (pET 28b was used as the vector) to the 33 identified amino acids were synthesized using site-directed mutagenesis by GenScript. Recombinant protein expression was carried out using the same protocols as the wild-type allergens.

Immuno dot blot assay

IgE binding assays were done using immuno-dot blots assays as previously reported.^6,8 Briefly, recombinant allergens (wild type or mutants, 0.5 mg/mL), were stamped onto 0.2 μm pore-size nitrocellulose membrane (Cytiva Life Sciences, UK) using a 384-pin Multi-Blot replicator (V&P Scientific Inc. VP386). Purified bovine serum albumin (BSA) and 1xPBS were used as background controls. Membranes were air-dried at room temperature prior to blocking, and assessment of specific IgE binding. The IgE-binding wild-type allergens or Der p 22 mutants were done using sera from atopic individuals, with positive SPT reactions to dust mite extracts. Normalization of the intensities was performed by subtracting the background. Intensities greater than 2 SDs above the mean negative sera responses were considered positive. The amount of specific IgE to recombinant allergens was estimated by comparing with a standard curve constructed using the human serum IgE standard (75/502; National Institute for Biological Standards and Control (NIBSC), United Kingdom). The specific IgE reactivity to dust mite allergens was assayed using immuno-dot blot assays and levels were calculated based on an IgE standard curve (Supplementary Figure 1). A cut-off of 0.35 IU/mL which is equivalent to ImmunoCAP class 1 was used as the cut-off for specific IgE reactivity on the immuno dot blot assay.^18,19 An alanine mutant was classified as a major IgE epitope if the IgE binding reduction was more than 80% compared to wild-type Der p 22 in at least half of the sera assessed.

Inhibition IgE binding assay

IgE Inhibition ELISA was carried out based on previously published protocols.^6,8 Briefly, Maxisorp 96-well microtiter plates (Nunc) were coated overnight with 250 ng recombinant allergen 100 mM bicarbonate/carbonate coating buffer (pH 9.6) per well overnight at 4 °C. Sera was pre-absorbed with varied amounts of recombinant protein (0.00001, 0.001, 0.001, 0.01, 0.1, 1.0, 10, 100 ng/μL) and incubated overnight at 4 °C. Plates were washed using PBS-0.05% Tween-20 and blocked using PBS-0.1% Tween-20. Pre-absorbed sera were added to each well overnight, followed by biotin-conjugated anti-human IgE monoclonal antibody (Sigma-Aldrich) and streptavidin HRP. The colorimetric reaction was detected by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate. The reaction was stopped using 1 M HCl, and absorbance was measured at 450 nm with a reference wavelength of 650 nm using an ELISA plate reader (Tecan). All the experiments were carried out in duplicates and results were reported as mean values with standard deviations. The degree of cross-reactivity was calculated by the percentage of inhibition based on the following formula:

[(Iu − Ii)/(Iu − B)] × 100%

where Iu indicates the reaction when the inhibitor protein was not present, Ii indicated the reaction at a particular inhibitor concentration, and B indicates the background intensity when the allergen was incubated with blocking solution in the absence of sera.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism (v 9.0). Spearman's correlation test was used to calculate the correlation of IgE reaction between allergens at a 95% confidence interval. The chi-square test or Fisher's exact test was used to compare the frequencies of 2 possible outcomes. The comparisons were two-tailed and the Mann-Whitney test was performed to compare the median of 2 possible outcomes. Multivariate logistic regression (adjusted for age and gender) was used to evaluate associations between specific IgE reactivity and clinical outcomes, including asthma and atopic comorbidities, in the combined Malaysian young adult and SMCGES cohorts. A P value < 0.05 was defined as statistically significant.

Results

Expression and purification of recombinant Der p 22 protein

The group 22 allergen of the dust mite D. pteronyssinus, Der p 22, consists of 492 nucleotides that encode 163 amino acid residues (Accession number A0A291KZA0,Supplementary Figure 2). Der p 22 contains a signal peptide cleavage site between amino acid residues 27 and 28, VQA-NDE as predicted by the SignalP algorithm. The DNA sequence of Der p 22 corresponding to the mature protein was synthesized with a hexa-histidine tag and expressed in E. coli strain BL21 (DE3). The resulting recombinant protein, with an estimated molecular weight of 17.2 kDa, was purified using nickel affinity chromatography (Supplementary Figure 3).

Participant characteristics and frequency of IgE binding to Der p 22

Participants from a cross-sectional cohort consisting of 176 Malaysian adults were assessed for their sensitization to common environmental allergen extracts (dust mites D. pteronyssinus and B. tropicalis, cockroach, oil palm pollen and fungus) to determine their atopic status (Table 1). Among the 176 individuals tested, 149 showed positive skin reactions to at least 1 of the allergens tested. Sera from the 149 atopic individuals were further analyzed for their IgE binding frequency to recombinant wild-type Der p 22 to determine the case and control groups (Supplementary Figure 4). The Der p 22-sIgE positive (n = 80) and negative individuals (n = 69) were comparable concerning the distribution of gender, mean age, body mass index (BMI), household income, and allergic disease presentation (Table 1).

Table 1.

Demographic data of Der p 22-positive and negative individuals in the atopic Malaysian adult cohort

Variables	Der p 22-sIgE positive individuals	Der p 22-sIgE negative individuals
Total (n)	80	69
Gender
Male (n, %)	19 (23.8)	21 (30.4)
Female (n, %)	61 (76.3)	48 (69.6)
Age (Year, mean ± SD)	23.16 ± 6.2	23.45 ± 7.5
BMI	21.91 ± 5.0	21.95 ± 5.2
Skin prick test positivea
D. pteronyssinus	67 (83.8)	67 (97.1)∗
B. tropicalis	60 (75.0)	59 (85.5)
P. americana	30 (37.5)	27 (39.1)
B. germanica	19 (23.8)	22 (31.9)
Monthly family income
< RM3000	20 (25.0)	9 (13.0)
RM3000-RM5999	43 (53.8)	48 (69.6)
>RM6000	11 (13.8)	10 (14.5)
Not indicated (n, %)	6(7.5)	2 (2.9)
Asthma	15 (18.8)	15 (21.7)
Allergic rhinitis	71 (88.8)	57 (82.6)
Atopic dermatitis	34 (42.5)	31 (44.9)
Allergic morbidityb
Single allergic disease	36 (45.0%)	36 (52.0%)
≥2 allergic diseases	38 (48.0%)	32 (46.0%)

An independent t-test and chi-square test were used to calculate the p-values (∗p < 0.05, ∗∗p < 0.01).

The participants were classified into Der p 22-positive (having IgE reactivity ≥0.35 IU/mL to Der p 22, ImmunoCAP class 1) and negative individuals (having IgE reactivity <0.35 IU/mL to Der p 22) using the results obtained from immune dot blot.

Data is presented as number of patients (n) (with percentage, %). Monthly family income: per capita income, AS: asthma, AR: allergic rhinitis, AD: atopic dermatitis and not indicated: individuals who did not respond to the question.

^aThe skin prick test result for the allergens tested was classified as positive if the mean wheal diameter was ≥3 mm.

^bThe individuals were classified into single allergic diseases (AS or AR or AD only), with ≥2 allergic diseases (AS and AR, or AS and AD, or AS, AR and AD).

Fifty-four percent of the atopic sera tested showed specific IgE binding to recombinant Der p 22 (Fig. 1). The same sera were tested for IgE binding frequency to other closely related allergens, namely Der p 2, Der f 2, and Der f 22. The majority of Malaysian adult atopic individuals demonstrated IgE reactivity to Der p 2 (97%) followed by Der f 2 (75%), and Der f 22 (34%). Individuals in the control group did not have serum-specific IgE to any of the recombinant allergens tested. As more than 50% of our study cohort had serum-specific IgE against Der p 22, it is classified as a major dust mite allergen.

Fig. 1.

Allergen sensitization profile of group 22 and group 2 allergens from D. pteronyssinus and D. farinae in the Malaysian population. Serum-specific immunoglobulin E (sIgE) titres (as IU/mL) against each allergen are indicated, using a cohort of 176 Malaysian adults. sIgE titres were assessed using immuno-dot blots. The sIgE reactivity was also classified based on ImmunoCAP classes. An IgE titer of <0.35 IU/mL was considered negative. The percentage of individuals with positive IgE reactivity is listed for each recombinant allergen

To further investigate the specificity of IgE responses to Dermatophagoides group 22 allergens, we analyzed a larger dataset (n = 755) from the SMCGES sub-cohort. The study methodology and detailed cohort demographics have been reported previously.20, 21, 22 Participants in the SMCGES sub-cohort exhibit demographic characteristics comparable to those of the Malaysian adult cohort (n = 176) (Supplementary Table 1). Within the SMCGES sub-cohort, 66.5% of individuals exhibited detectable specific IgE responses (>0.35 IU/mL) to Dermatophagoides crude allergens (Supplementary Table 1). Among these crude allergen responders, 79.7% and 45.6% had detectable specific IgE responses to group 2 and group 22 allergens, respectively (Supplementary Fig. 5A and B). Notably, 35 individuals (6.8% of crude responders) exhibited specific IgE responses to group 22 allergens in the absence of detectable group 2-specific IgE (Supplementary Figure 5C). Furthermore, in the full SMCGES sub-cohort (n = 755), specific IgE titers to group 2 and group 22 allergens were not correlated (Pearson's R = −0.00615), whereas a strong correlation was observed between IgE titers to Der p 2 and Der f 2 (Pearson's R = 0.568,Supplementary Fig. 5D and E). Collectively, these findings suggest the presence of monosensitization to Der p 22 within this study population.

Association between IgE reactivity to Der p 22 and corresponding clinical outcomes

The correlation between sIgE reactivity to Der p 22 and clinical outcomes regarding allergic disease presentation and their symptoms were further assessed. Atopic individuals with Der p 22 sIgE had a higher tendency to develop allergic rhinitis compared to those without Der p 22 sIgE (P = 0.008,Supplementary Table 2). Further analysis of allergic rhinitis symptoms revealed that individuals with Der p 22-sIgE were more prone to experience intermittent blocked nose, runny nose, and sneezing than individuals without Der p 22-sIgE (Fig. 2). No significant differences were observed in our patient cohort for asthma exacerbation outcomes (wheezing, asthma attacks, school absence, and A&E admissions) between the Der p 22-sIgE positive or negative individuals, due to limitations in sample size (Supplementary Table 3). By combining the Malaysian adult cohort (n = 176) with the SMCGES sub-cohort (n = 755) and performing multivariate logistic regression analysis (age- and gender-adjusted), we found that a Class 3 or higher specific IgE response (>3.5 IU/mL) to group 22 allergens was associated with an increased risk of asthma in this population (p < 0.0524, OR = 1.73, 95% CI = 0.98–3.00,Supplementary Figure 6A). Group 22-specific IgE responses (Class 3+) were also significantly associated with atopic comorbidities involving asthma, allergic rhinitis (AR), and atopic dermatitis (AD) (all p < 0.05,Supplementary Figure 6A). Notably, these associations remained significant among individuals who also exhibited specific IgE responses against group 2 allergens (all p < 0.05,Supplementary Figure 6B). Overall, these findings supported the clinical relevance of group 22 IgE reactivity, which appears to be independent of group 2 IgE reactivity in the Singapore/Malaysia population.

Fig. 2.

Allergic rhinitis symptoms among Der p 22-sIgE positive and negative Individuals. Atopic individuals were classified as Der p 22-positive or -negative based on their IgE-reactivity to wild-type Der p 22 using immuno-dot blots. Differences in the percentage of Der p 22-positive or negative individuals suffering from allergic rhinitis symptoms such as blocked nose, runny nose, sneezing or itchy nose were determined based on the responses to the standardized survey questionnaire and assessed using Chi-squared test or Fisher's exact test (∗p < 0.05 and ∗∗p < 0.01)

Der p 22 harbours unique IgE-epitopes

The correlation of direct IgE-binding to Der p 22 and its structural homologue Der p 2 demonstrated a moderate positive correlation which was statistically significant (r = 0.562, P < 0.001) (Supplementary Figure 7). To further delineate if the positive correlation observed was due to co-sensitization or cross-reactivity between both allergens, an inhibition ELISA was carried out. Sera from 3 individuals, MI23, MI183, and MI337, each exhibiting unique IgE reactivity profiles to Der p 22 and Der p 2 were evaluated (Supplementary Figure 7). MI23 exhibited high IgE reactivity to both allergens (>1000 IU/mL), MI183 showed moderate IgE reactivity to Der p 2 (10IU/mL), but low reactivity to Der p 22 (0.5IU/mL), while MI337 displayed moderate IgE reactivity to both Der p 22 and Der p 2 (5–8 IU/mL). In all 3 sera tested, Der p 22 was partially cross-reactivity to Der p 2 (58%–75%), indicating the presence of unique IgE epitopes on Der p 22 (Fig. 3).

Fig. 3.

IgE inhibition assays between Der p 22 and Der p 2. Dose-dependent inhibition of IgE binding to surface-coated Der p 2 or Der p 22 was performed in the presence of increasing concentrations of Der p 2 or Der p 22 allergens in ELISA. Three individual sera (MI23, MI183, and MI337) with different IgE-binding profiles to Der p 22 and Der p 2 were assayed for self- and cross-inhibitions to both allergens

Identification and characterization of IgE-binding epitopes of Der p 22

Next, we sought to identify and characterize the IgE-binding epitopes of Der p 22. Based on previous studies, allergen IgE-binding epitopes mainly consist of surface-exposed polar and charged amino acid residues.^4,8,13, 14, 15, 16, 17 A predicted three-dimensional model of Der p 22, based on the solved X-ray structure of Der f 2 (PDB: 1XWV.1.A), was used to identify putative IgE-epitope residues based on their solvent accessibilities. Thirty-three surface-exposed polar and charged residues of Der p 22 were selected to produce a set of 33 single alanine mutants (Supplementary Table 4). The single-alanine mutants were expressed and purified using the same methods as the wild-type Der p 22 (Supplementary Figure 2).

Next, the amino acid residues important in the IgE reactivity of Der p 22 were determined by analysing the impact of each alanine mutation on IgE binding in comparison to wild-type Der p 22. Amino acid residues were considered to significantly contribute to IgE binding if their alanine mutants exhibited a binding reduction of over 80% compared to the wild-type Der p 22 in at least half of the sera tested. Out of the 33 amino acid residues assessed, eleven demonstrated a significant reduction in IgE-binding when compared to the wild-type allergen. These 11 residues, K10, H28, K30, H35, R36, K39, R43, D106, R117, R130, and H132A, were identified as major IgE epitopes of Der p 22 (Fig. 4A, left panel). Topographically, these IgE-binding residues were distributed evenly across the predicted molecular surface of Der f 22 (Fig. 4A, right panel). Alanine mutation of residue D106 resulted in the most profound reduction in IgE binding, with 73 of the 80 sera (91%) tested showing more than 80% reduction in IgE binding; followed by K10 (72 sera; 90%), R130 (71 sera; 90%), H53 (70 sera; 87.5%) and R43 and R117 (both 66 sera; 66%). Of the remaining 22 mutants, 15 had reductions in IgE-binding ranging from 10% to 50%. The remaining 7 mutants, E4, D11, E68, D75, D78, E119 and D120, did not demonstrate any reduction in IgE reactivity when compared to wild-type Der p 22 (Fig. 4A).

Fig. 4.

Determination of the major IgE epitopes of Der p 22. Site-directed mutagenesis was performed on selected surface residues of Der p 22 to alanine, and the resulting mutants were assayed for their IgE-binding capacity compared to the wild-type allergen. Data was depicted for (A) the whole population (n = 80) and (B) according to the specific IgE titre to Der p 22 (top panel: CAP 1, middle panel: CAP 2, bottom panel: CAP 3–6). Mutations resulting in more than 80% reduction in IgE binding in each serum were considered significant (highlighted black). (C) Nonparametric Spearman correlation analysis of the number of IgE binding residues and the Der p 22-sIgE titers show a statistically significant positive correlation (Spearman correlation coefficient, r = 0.407, P < 0.001)

Next, we evaluated the effects of sIgE titres on the level of Der p 22 epitope IgE reactivity. Eighty sera having different IgE titres to Der p 22 were classified into 3 groups based on ImmunoCAP; low sIgE level (CAP class 1, n = 15), moderate sIgE level (CAP class 2, n = 32) and high IgE levels (CAP classes 3–6, n = 33). The number of IgE Der p 22 mutants contributing significantly to a reduction in IgE binding increased progressively from low (n = 5) to moderate (n = 10) to high (n = 15) (Fig. 4B). This observation was further supported by the statistically significant positive correlation (r = 0.407, P < 0.001) between the number of IgE binding residues and Der p 22-sIgE titres (Fig. 4C), indicating that individuals with higher Der p 22-sIgE levels recognized a greater number of Der p 22 IgE epitopes.

The IgE-reactivity patterns of individual alanine mutants of Der p 22 were further examined to better understand epitope-specific responses at the individual patient level (Fig. 5). Each mutant displayed a distinct IgE-binding signature within the tested population. One of the outcomes of understanding the IgE epitope profile of an allergen is to design hypoallergenic variants for use Mapping epitope profiles provide a foundation for the future development of modified allergen preparations with reduced IgE-binding capacity.

Fig. 5.

Profiling of major Der p 22 IgE epitopes reveals distinct IgE-binding patterns. Major IgE epitopes of Der p 22 were profiled for their IgE binding capacity among 80 atopic Malaysian adults with IgE reactivity to wild-type Der p 22. Each column in the heatmap represents an individual Der p 22 alanine mutant tested, and each row represents the percentage of reduction of IgE binding to the mutant to individual atopic individuals, as compared to the wild-type allergen (0–100%)

Given the limitations of conventional allergen extracts used in AIT, including the risk of IgE-mediated adverse reactions,^23,24 strategies that incorporate hypoallergenic variants have been proposed as a potentially safer alternative. In this study, alanine mutations to 6 residues, K10, H35, R43, D106, R117 and R130 led to a marked decrease in IgE binding (median reduction >95%), suggesting these residues play a key role in IgE recognition (Fig. 5).

Polysensitized individuals recognized more Der p 22 IgE-binding residues compared to those sensitized only to dust mites

We next evaluated whether the allergic sensitization profile of an individual influenced their IgE epitope recognition. To assess this, sera (n = 75) of our study cohort was divided into 2 groups based on their skin reactivity to common tropical allergen extracts. The first group comprised individuals with skin reactivity to dust mite (DM) extracts only (n = 29), while the second group comprised individuals with skin reactivity to allergens from various sources (polysensitized to 2 or more of the following sources: DM, cockroaches, oil palm pollen or fungus; n = 46). As shown in Fig. 6A, the number of epitopes recognized in the polysensitised group (13 epitopes) was higher than in the dust mite-sensitized group (11 epitopes).

Fig. 6.

Analysis of the major IgE-epitopes of Der p 22 based on clinical parameters. Atopic individuals were classified based on their (A) sensitization profiles to allergen extracts (sensitized to dust mites D. pteronyssinus and/or B. tropicalis vs polysensitized to dust mites and/or cockroaches (P. americana and B. germanica) and/or oil palm pollen (E. guineensis) and/or fungus (Curvularia spp.), (B) allergic multimorbidity (single allergic disease vs poly-allergic), or (C) severity of allergic rhinitis symptoms (mild vs moderate-to-severe). Major IgE binding epitopes (highlighted black) are those with >80% reduction in IgE binding in mutants compared to wild-type Der p 22 in at least half of the population tested were depicted

Individuals with multiple allergic diseases recognized more Der p 22 IgE epitopes compared to individuals with a single allergic condition

A study by Pang et al. in 2021 reported that individuals presenting with 2 or more allergic diseases (poly-allergic) recognized more IgE epitopes compared to those with only a single allergic disease.⁴ We assessed if the same trends were observed in our study cohort regarding Der p 22 IgE epitope recognition. Atopic individuals with sIgE to Der p 22 were classified into those having a single allergic disease (n = 36) versus those with 2 or more allergic diseases (AS and AR, AS and AD, AR and AD, AS, AR and AD; n = 38, termed poly-allergic). The number of Der p 22 IgE epitopes recognized in the poly-allergic (n = 20) was significantly higher compared to that of the single allergic group (n = 11) as shown in Fig. 6B.

Effects AR severity on the number of Der p 22 IgE epitope recognized

We further assessed if the IgE epitope recognition on Der p 22 was influenced by the severity of AR among Malaysian atopic adults. Individuals were classified as having mild AR (n = 28) or moderate-to-severe AR (n = 43) based on their responses to a validated survey questionnaire regarding their allergic symptoms. We found that individuals with moderate-to-severe AR recognized slightly more IgE epitopes of Der p 22, compared to the group with mild AR (12 vs 11 epitopes) as depicted in Fig. 6C.

Discussion

Dermatophagoides spp. are the most significant allergy-causing dust mites (DM) worldwide, owing to their ability to adapt to different climatic conditions.^2,3 It causes IgE-mediated allergic reactions in both paediatric and adult allergic populations.^6,8,25 In fact, in this study, 72% of a cross-sectional Malaysian adult population was sensitized to Dermatophagoides pteronyssinus (DP) and D. farinae (DF) based on skin prick reactivity to its allergen extracts, consistent with previous reports.25, 26, 27, 28, 29, 30 Additionally, our epidemiological survey data demonstrate a significant association between dust mite sensitization and allergic disease presentation, similar to previously published studies.31, 32, 33, 34, 35 Detailed characterization is not available for all DM allergens. In this study, we characterize the 17.2 kDa Der p 22 in terms of its IgE binding profile, immunoglobulin E (IgE) epitopes and clinical significance of allergic disease presentation among Malaysian atopic adults.

Der p 22 is the group 22 allergen of DP, and a structural homologue of the well-studied group 2 allergen from Dermatophagoides spp. Hence, in this study, we compared the IgE reactivity of our adult atopic population to recombinant group 22 and group 2 allergens from DP and DF. Der p 22 was a major allergen within our population, with 54% of the sera tested demonstrating specific IgE (sIgE) reactivity. We additionally observed high IgE reactivity to Der p 2 (97%) and Der f 2 (75%) echoing previous findings.^6,25,36,37 Interestingly, the sIgE reactivity to Der p 22 did not correlate with skin prick test (SPT) reactivity using DP extracts. This could be due to the under-representation of this allergen in the extract due to the preparation method, naturally present at low concentrations, or allergen degradation.³⁸ These observations suggest that diagnosis with allergen extracts alone is insufficient to detect Der p 22 sensitization. Other methods, such as component-resolved diagnosis should be considered for improved allergen diagnosis accuracy.³⁹

Sensitization to Der p 22 demonstrated a statistically significant association with the presence of allergic rhinitis symptoms in Malaysian adults, suggesting its role as a clinically relevant allergen in this population. The observed correlation between Der p 22-specific IgE responses and symptomatic disease underscores the allergen's potential contribution to disease pathogenesis beyond immunological sensitization alone. These findings suggest the importance of including Der p 22 into component-resolved diagnostic platforms to improve the precision of allergy diagnostics and underscore its importance as a target for precision allergen-specific immunotherapy in sensitized populations.

Although Der p 22 is a structural homologue of Der p 2, it possesses unique IgE epitopes. Using alanine mutation studies, a detailed IgE epitope characterization of Der p 22 revealed the presence of 11 major IgE epitopes, namely K10, H28, K30, H35, R36, K39, R43, D106, R117, R130, and H132. These IgE epitopes were distributed randomly on the modelled three-dimensional structure of the Der p 22 allergen (Supplementary Figure 8), consistent with the characteristics of IgE epitopes of other allergens.^4,16,40, 41, 42, 43 While each of the immunodominant epitopes showed unique IgE reactivity profiles, for 3 mutants, K10, D106, and R130, the reduction in IgE binding was observed in the majority (>90%) of the sera tested. Further characterization of these alanine mutants using functional assays such as basophil activation test or histamine release assays would provide additional confirmation on the reduced allergenic activity of the hypoallergenic mutants of Der p 22. In addition, characterization of these mutants in suitable animal models will be necessary to determine their suitability as candidate hypoallergen vaccines against Der p 22 allergies.

In our study population, a positive correlation between IgE epitope diversity (ie the number of epitopes recognized) and the titre of specific Der p 22 IgE was observed, similar to previous findings of other dust mite allergens such as Der f 21 and Der p 23.^4,16 Der p 22 sIgE reactions were found to be more pronounced in individuals with sensitizations to multiple allergen sources. This suggests that polysensitized individuals have a wider recognition of allergen epitopes, as reported previously for dust mites,^4,16 pollen,⁴⁴ and food allergens,^45,46 which could potentially lead to more severe or persistent allergic symptoms. The broad recognition of allergens and epitopes further underscores the importance of precision and personalized medicine, where treatments are tailored based on the specific needs of the patient, which can effectively desensitize the immune system. This knowledge could be translated into the development of hypoallergenic molecules for AIT or monoclonal antibodies for specific biological treatments.⁴⁷ More research on the efficacy of personalized component-resolved immunotherapy over conventional extract-based preparations to address the individual needs of allergic individuals is necessary moving forward.

Availability of data and materials

All data used and included in this study are available from the corresponding author (K.R.).

Author contributions

K.R. conceived and supervised the current research study. S.M.R.S. conducted the literature review, recruited study participants, analyzed and interpreted the data, and wrote the manuscript. A.S.L.L. and S.Y.Y. assisted in recruitment of study participants, data analysis and drafting of the article. C.F.T. supported data interpretation and analysis. All authors read and approved the final manuscript.

Ethics approval and consent

Ethical approval for this study was obtained from the Sunway University Ethics Committee (Reference – SUREC2019/029). This study was performed in compliance with the Declaration of Helsinki, Good Clinical Practice, and local regulatory guidelines. Before participating, each subject was informed of this study's details via a Participant Information Sheet and provided written informed consent to participation through the signature of a Consent Form.

Authors’ consent for publication

All authors have read and consented to the publication of this manuscript.

Declaration of Generative AI and AI-assisted technologies in the writing process

ChatGPT was used to improve readability and language of this manuscript.

Funding

K.R. has received research supported by the Fundamental Research Grant Scheme (Project code FRGS/1/2021/SKK0/SYUC/02/1) awarded by the Ministry of Higher Education Malaysia and Sunway University Research Grants (GRTIN-IGS(02)-DBS-09-2022), and Sunway University Collaborative Research Fund: T20 (STR-RMF-T20-005-2019). F.T.C. received grants from the National University of Singapore (N-154-000-038-001 (E−154-00-0017-01); C141-000-077-001 (E−141-00-0096-01)), Singapore Ministry of Education Academic Research Fund (R-154-000-191-112; R-154-000-404-112; R-154-000-553-112; R-154-000-565-112; R-154-000-630-112; R-154-000-A08-592; R-154-000-A27-597; R-154-000-A91-592; R-154-000-A95-592; R154-000-B99-114), Biomedical Research Council (BMRC) (Singapore) (BMRC/01/1/21/18/077; BMRC/04/1/21/19/315; BMRC/APG2013/108), Singapore Immunology Network (SIgN-06-006; SIgN-08-020), National Medical Research Council (NMRC) (Singapore) (NMRC/1150/2008; OFIRG20nov-0033; MOH-001636 (OFLCG23may-0038, A-8002641-00-00)), National Research Foundation (NRF) (Singapore) (NRF-MP-2020-0004), Singapore Food Agency (SFA) (SFS_RND_SUFP_001_04; W22W3D0006), Singapore's Economic Development Board (EDB) (A-8002576-00-00), and the Agency for Science Technology and Research (A∗STAR) (Singapore) (H17/01/a0/008; and APG2013/108). This research is supported by the National Research Foundation Singapore under its Open Fund-Large Collaborative Grant (MOH-001636) (A-8002641-00-00) and administered by the Singapore Ministry of Health's National Medical Research Council. The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of competing interest

No competing interests disclosed.

Appendix A. Supplementary data

The following is the Supplementary data to this article.