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Variation in IgE binding potencies of seven Artemisia species depending on content of major allergens

Variation in IgE binding potencies of seven Artemisia species depending on content of major... Background: Artemisia weed pollen allergy is important in the northern hemisphere. While over 350 species of this genus have been recorded, there has been no full investigation into whether different species may affect the allergen diagnosis and treatment. This study aimed to evaluate the variations in amino acid sequences and the content of major allergens, and how these affect specific IgE binding capacity in representative Artemisia species. Methods: Six representative Artemisia species from China and Artemisia vulgaris from Europe were used to deter- mine allergen amino acid sequences by transcriptome, gene sequencing and mass spectrometry of the purified allergen component proteins. Sandwich ELISAs were developed and applied for Art v 1, Art v 2 and Art v 3 allergen quantification in different species. Aqueous pollen extracts and purified allergen components were used to assess IgE binding by ELISA and ImmunoCAP with mugwort allergic patient serum pools and individual sera from five areas in China. Results: The Art v 1 and Art v 2 homologous allergen sequences in the seven Artemisia species were highly con- served. Art v 3 type allergens in A. annua and A. sieversiana were more divergent compared to A. argyi and A. vulgaris. The allergen content of Art v 1 group in the seven extracts ranged from 3.4% to 7.1%, that of Art v 2 from 1.0% to 3.6%, and Art v 3 from 0.3% to 10.5%. The highest IgE binding potency for most Chinese Artemisia allergy patients was with A. annua pollen extract, followed by A. vulgaris and A. argyi, with A. sieversiana significantly lower. Natural Art v 1-3 isoallergens from different species have almost equivalent IgE binding capacity in Artemisia allergic patients from China. Conclusion and clinical relevance: There was high sequence similarity but different content of the three group allergens from different Artemisia species. Choice of Artemisia annua and A. argyi pollen source for diagnosis and immunotherapy is recommended in China. Keywords: Pollen allergen, Artemisia, Different species, IgE binding potency, Allergen quantification Background Artemisia species are wind-pollinated weeds, widely dis- tributed in the northern hemisphere with a few species *Correspondence: gaozhongshan@zju.edu.cn † in the southern hemisphere [1]. Pollens of Artemisia have Lan Zhao and Wanyi Fu should be considered joint first author Allergy Research Center, Zhejiang University, Hangzhou 310058, China been recognized as a major cause of late summer and Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhao et al. Clin Transl Allergy (2020) 10:50 Page 2 of 13 autumn seasonal allergic respiratory disease, especially be better for immunotherapy (Bai et  al. China Patent, along the Asia-Europe silk-road and in north-western CN102512673B). With a serum pool from the USA, high United States [1–6]. Between 350 and 500 Artemisia levels of cross-reactivity has been found with ELISA species have been recorded in the plant kingdom [1, 7] inhibition in nine Artemisia species, with two local sage worldwide, 187 in China [8]. Phylogeny of the Artemisia species being the strongest inhibitors [1]. Very recently, genus, updated by molecular marker analysis [7, 9], has using immunoblots, similar IgE binding patterns of seven reached a consensus of six sections: Artemisia, Abro- Artemisia species have been found, with some degree of tanum, Dracunculus, Absinthium, Seriphidium and difference in three major allergen bands [17]. Tridentata. Most Artemisia species are in the first four Cross-reactivity has been found in different Artemisia sections and are distributed in temperate climate regions, species [1], but whether different species in China have where the majority of mugwort pollen allergic patients an impact on the allergen diagnosis and treatment has live. The few species belonging to Seriphidium and Tri - not been fully investigated. This study aims to provide a dentata are distributed in semi-desertic to steppic envi- comprehensive analysis of sequence variation of different ronments [10]. Some Artemisia species are dominant in isoforms and variants, content of allergens Art v 1, Art v natural plantations, contributing to the geographic dif- 2 and Art v 3, and their impact on IgE binding of six rep- ference of the pollen allergy [5]. Artemisia vulgaris is the resentative Artemisia species in China. best studied species, mainly distributed in northwestern and central Europe. Five major species have been listed Materials and methods in China (A. annua, argyi, sieversiana, capillaris, lavan- A graphic research design is presented in Fig.  1, with dulifolia) in a national pollen survey [11], and there is detailed information given in the following sections. preliminary clinical and immunological evidence of the potential IgE binding potency of the first three species Artemisia species and protein extract [12, 13]. A few species, such as A. annua, have invaded We used pollens of seven Artemisia species: five domi - Europe and America, becoming potentially severe aller- nant in Northern China (A. annua, A. argyi, A. capillaris, genic sources [14]. Artemisia pollen allergy is directly A. sieversiana, A. gmelinii) collected from Shanxi Prov- related to the distribution of Artemisia spp., density, ince; A. lavandulifolia from Zhejiang Province in South climate [6] and risk factors [15]. Currently, commercial China collected over three years (Additional file  1: Fig- mugwort pollen allergen extract CAPs are from A. absin- ure S1), and Artemisia vulgaris pollen purchased from thium (w5) and A. vulgaris (w6), the latter being the most Allergon in Sweden. Species authenticity was verified by commonly used in diagnosis. cloning and sequencing the ITS2 on genomic DNA from Molecular characterization of Artemisia vulgaris and pollen according to a reported method [24]. Aqueous Artemisia annua has revealed seven allergens, with the protein extracts of pollen were prepared by resuspend- clinical data and reference DNA and protein sequences ing 0.2 g pollen grains in 3.5 ml PBS or 2 g in 35 ml PBS published [16, 17]. Art v 1 and Art v 3 have been shown buffer (0.14  M NaCl, 2.7  mM KCl, 7.8  mM Na HPO , 2 4 to be major allergens worldwide, and a newly identified 1.5 mM KH PO ), and shaking for 12 h at 4 °C. Extracts 2 4 group, Art an 7 also seems to be important, although were centrifuged at 10,000 g for 10  min at 4  °C, filtered its IgE values are usually much lower [3, 18–20]. By through 0.22  µm filters (Millipore), and the concentra - sequence cloning of a single species of Artemisia vulgaris tion of protein in extracts was determined using the BCA pollen, seven Art v 1 isoforms have been identified, with protein assay kit (Takara Bio, Japan). Three independent only slight variation in the C-terminal and very similar extracts from different pollen samples of each species IgE reactivity [21]. Five Art v 3 isoforms have also been from China, and one A. vulgaris sample were prepared identified, one a partial sequence by N-terminal sequenc - and used in whole protein and individual allergen quan- ing [22] and the other four by gene cloning [23]. Diver- tification. The rArt v 1.0101 and rArt v 3.0201 standards sity of group 7 allergen sequences of seven Artemisia were from previous studies [21, 23]. species has recently been reported, where two isoforms for each species have been found with over 95% identical RNA extraction and Transcriptome sequence [17]. Total RNA was extracted from pollens of six Artemisia The current commercial mugwort pollen extract used spp., collected from China, using the RNAprep pure kit for skin prick and immunotherapy in China is mainly (Tiangen, China) [17], and sequenced by BGI-Shenzhen from A. sieversiana,  even though A. annua was rec- and Hangzhou One Gene Ltd using Illumina HiSeq ognized as an important allergen source in the 1980  s 2000 (San Diego, CA, USA). De novo transcriptome [5], and a recent report states that a mixture of pollens assembly was using the Trinity software package with from three species (argyi, annua, sieversiana) would a minimum K-mer of 3 and a minimum contig size of Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 3 of 13 Fig. 1 Overview of research design and outcomes 100  bp. After extraction of allergenic protein sequences, AS-LTP-Rev: TCA TTT CAC CTT GTT GCA ATC. PCR −5 blastx was used for alignment (e-value, 1e ) between amplification with both primer pairs was using Phusion Unigenes and protein databases from the Artemisia High-Fidelity DNA Polymerase (New England Biolabs, annua genome [25]. USA). At least eight clones were selected for sequenc- ing. Nucleotide sequences and deduced amino acid Cloning of Art v 1, Art v 2 and Art v 3 homologues sequences from different pollens have been deposited in Pollen cDNA was prepared with the PrimeScript   RT GenBank. Isoallergens and variants were named follow- reagent kit with gDNA Eraser (Takara Bio, Japan) using ing the nomenclature and the updated official list of the mRNA fragments as templates. The full length of Art WHO/IUIS Allergen Nomenclature Sub-committee [26] v 1, Art v 2 and Art v 3 homologues were obtained by and have been approved. PCR using primers based on Art v 1.0101 (AF493943) sequences: Art v 1-For, 5´-AAT GGC AAA GTG TTC Natural allergen purification and protein identity ATA TGTT-3´, Art v 1 - Rev, 5´-TTA GTG AGT GGA Monoclonal antibodies (mAb) used in this study were CGG AGG AG-3´; Art v 2 sequence (AM279693) Art v from previous research [27]. A7-G4-E6 specific to Art 2-For, CCT CAT ACA GAA ACA TGG GAC, Art v 2-Rev, v 1, C9-C1 to Art v 2, and A2-B8 to Art v 3, were used TTA GTA AGG TTT CTG ACC AAC AAC; and Art v 3.02 to purify three groups of allergens from six selected (EU564845, EU454846 original sequences provided by Artemisia spp. from China as described previously [27]. Gabriele Gadermaier) Art v 3-For, 5´-ATG GCA ATG LC–MS/MS (Thermo Scientific Q Exactive) was used AAA ATGA TGA A-3´ and Art v 3- Rev, 5´-CTA GCA for identity-matching of the purified proteins to deduced TAA AGY AYT TCA C-3´for first round cloning. Accord - allergens from each species. The purity of natural aller - ing to newly assembled transcript sequences for Art v 3 gens was estimated by SDS-PAGE. group, additional cloning for A. capillaris with AC-LTP- For: ATG GCA ATR AAA ATG ATG AAGG, AC-LTP-Rev: Quantification of three components by ELISA TTC CAT GTA TTC CAG CAT AAA; and A. sieversiana A. argyi extract was used to immunize two New Zealand with AS-LTP-For: ATG GCA ATG AAA ATG ATG AAG, rabbits to produce polyclonal antibodies (pAb), injecting Zhao et al. Clin Transl Allergy (2020) 10:50 Page 4 of 13 with 500 μg protein in incomplete Freund’s adjuvant, fol- Chinese Artemisia species was assessed by ELISA, using lowed by three subcutaneous boosters of 250 μg protein sera of 142 individual mugwort-allergic patients. ELISA at intervals of 7-14  days. The quality was checked using plates (Corning, USA) were coated with 0.5 μg/well pol- both Western blot and ELISA. The antibodies were pro - len extracts in PBS buffer (pH 8.3). After blocking with duced by Hua An Biotech Ltd., Hangzhou, China. An 100  μl 5% skimmed milk, 100  μl serum pool was added, mAb (A7-G4-E6) and rabbit pAbs were used for quanti- with a negative serum pool as control. After washing, fication of Art v 1 homologous proteins. A mAb (C9-C1) 100 μl goat anti-human IgE coupled with HRP (1:3000 in and rabbit pAbs were used for quantification of Art v 2 PBS buffer) was added and bound IgE was detected using homologous proteins. A selected mAb pair (A2-B8 and TMB. The ELISA was quantified using the colorimetric biotinylated A9-G10) ELISA assay was used to quantify reaction at 450/620 nm. We also compared the IgE bind- Art v 3 homologous proteins with different recombi - ing values with rArt v 1.0101 and rArt v 3.0201 allergens nant or natural allergen standards. ELISA plates (Corn- tested by ELISA and the values of Art v 1 and Art v 3 ing, USA) were coated with 0.3  μg capture antibodies sIgE tested by ImmunoCAP. For the patients who were (A7-G4-E6, C9-C1, A2-B8) at 4 °C overnight, after block- positive in ImmunoCAP with A. vulgaris but negative in ing with 100 μL 5% skimmed milk at 37  °C for 1  h, 100 ELISA with the extracts of six Chinese Artemisia spp., μL serially diluted allergen standards and pollen extracts IgE binding capacity was further tested using a mixture were added and incubated at 37°C for 1  h. After wash- of pollen extracts of A. annua, A. argyi, and natural puri- ing, the wells were incubated with 0.3  μg biotinylated fied Art an 3 and Art ar 3 in a mass ratio of 4:4:1:1 (total detection antibodies at 37 °C for 1 h followed by incuba- of 0.5 μg/well mixture). tion with 100 μL HRP-conjugated Streptavidin (1:5000 Inhibition curves were obtained using inhibitors with dilution) at 37  °C for 1  h. Finally, 100 μL TMB (3, 3′, 5, serial dilutions of pollen extracts and recombinant A. 5′-tetramethylbenzidine) was added as colorimetric sub- vulgaris allergens in competition with a solid phase strate, and after incubation in the dark for 10  min, the coated with rArt v 1.0101 and rArt v 3.0201 for IgE bind- reaction was stopped by adding 50 μL 2 M HCl. The opti - ing, using the serum pools from Shanxi and Shandong. cal density was measured at 450/620  nm (MultSkan FC, ImmunoCAP inhibition on commercial mugwort (A. vul- Thermo Fisher, USA). For each species, the allergen con - garis) extract was with serial dilutions of pollen extracts tent was measured using three independent extracts with from three species (A. annua, A. sieversiana, A. vulgaris) duplicate wells. against individual serum from four groups of different sensitization patterns (Art v 1 and Art v 3 IgE positive or negative). Patients A total of 150 patients (Additional file  1: Table S1) allergic to mugwort were recruited from Datong-Shanxi (111); ImmunoCAP tests Taiyuan-Shanxi (11); Beijing (10); Yantai-Shandong (10), According to the sequence diversity, different natural and Qvjing-Yunnan (8) in China based on a convinc- purified allergens from the three groups were selected ing case history and positive IgE reactivity to mugwort for testing the IgE by ImmunoCAP. Allergens were bioti- extracts determined by ImmunoCAP (Thermo Fisher nylated and coupled to streptavidin‐conjugated Immu- Scientific, Uppsala, Sweden). Eighty-two of these patients noCAPs (Thermo Fisher Scientific, Uppsala, Sweden) have been reported previously [17, 20, 28]. Specific IgE to at 37 °C for 30 min and then were tested with sera of 18 the major mugwort allergen components, Art v 1 and Art individual mugwort-allergic patients. v 3 was determined by ImmunoCAP. Individual sera and serum pools from the five areas were used to assess IgE binding capacity. The sera of five non-atopic individuals Statistical analyses were pooled and used as a negative control. Written con- Data were analyzed by SPSS21.0, with a value of P < 0.05 sent was obtained from all participants (or their repre- considered significantly different. Graphs were drawn sentatives) and the study was approved by the local ethics with GraphPad Prism6.0. The ANOVA model with Tuk - committee. ey’s post hoc test was used to analyze the differences in protein content between seven Artemisia spp. Differ - ELISA binding and inhibition analyses ence in IgE reactivities was analyzed with the Friedman Pollen extracts from the seven species were used to ana- test and Dunn’s multiple comparison test. The Kruskal– lyze IgE binding by ELISA, with serum pools of patients Wallis test with Dunn’s test was used to determine the from the cities of Datong and Taiyuan in Shanxi prov- quantitative variables of the three allergen components, ince, Beijing, Yantai-Shandong and Qvjing-Yunnan. and Spearman’s correlation coefficient analysis to evalu - Further IgE reactivity of each pollen extract of the six ate correlations between ImmunoCAP scores and ELISA Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 5 of 13 values. The four-parameter dose–response curve models Table  S2). The natural allergens purified by mAb (Addi - were used to build the ELISA standard curves. tional file  1: Figure S2) were matched to the target aller- gen sequences, and no other allergens were found by Results mass spectrometry (Additional file  1: Figure S3). This Patients gave six new deduced defensin-like (Art v 1 type) pro- Among 150 patients (71 male, 79 female; age range teins in six Artemisia spp. in China, with six in the IUIS 6-62  years old, mean 30.9 ± 14.7  years), 137 (91%) were from reference A. vulgaris and the other five species diagnosed with allergic rhinitis, 43 (29%) with conjunc- (Fig.  2a). They are highly conserved at the N-terminus, tivitis, 42 (28%) with asthma and 12 (8%) with eczema. with seven variable amino acids (Fig.  2a). We identified For IgE reactivity against the major components, 103 a unique amino acid, 13  W, in the defensin-domain that (68.7%) were positive to Art v 1, 74 (49.3%) were positive was present in three of the species from China (A. annua, to Art v 3, and 61 (40.7%) were positive to both, while 24 A. capillaris, A. sieversiana) and in four American Arte- (16%) were negative to both allergens (Additional file  1: misia species [29], but not in A. vulgaris. In the proline Table S1). domain, there was 78S/P substitution in A. vulgaris, A. argyi and A. lavandulifolia, while in A. sieversiana, only Sequence variation of Art v 1, Art v 2 and Art v 3 78S was found, and in A. annua, A. capillaris and A. homologous proteins gmelinii only 78P. Three types of allergens in seven Artemisia spp. were One Art v 2 homologous isoform was obtained from identified by a joint analysis of pollen transcriptome each species, resulting in four different sequences from assembly, PCR cloning and sequencing (Additional file  1: seven species: Art v 2.0101 (MF326222), Art ar 2.0101 Fig. 2 Comparison of pollen allergen deduced amino acid sequences from different Artemisia species. a defensin-like protein (Art v 1 homologous allergen); b pathogenesis-related protein 1 (Art v 2 homologous allergen); c non-specific lipid transfer protein (Art v 3 homologous allergen). Underlined sequences indicate the allergens identified in previous research. GenBank accessions are listed in Additional file 1: Table S2 Zhao et al. Clin Transl Allergy (2020) 10:50 Page 6 of 13 and Art la 2.0101 were identical, as were Art gm 2.0101 ** and Art an 2.0101, and Art si 2.0101 had an isoform with *** *** two extra amino acids (Fig. 2b). The current reference Art ** *** A. annua * *** v 2.0101 in IUIS was deduced from AM279693, it was not A. argyi confirmed in this study. A. capillaris A. gmelinii More sequence variations were observed in the lipid A. lavandulifolia transfer proteins (Art v 3 type), with a total of nine iso- A. sieversiana forms or variants and up to 38 amino acids difference (Fig.  2c). Identical isoforms were found in different spe - cies. Two isoforms, Art an 3.0102 and Art si 3.0101, had a few specific amino acids (Fig.  2c). Most isoforms from the six Chinese Artemisa spp. were verified by mass spec - trometry after immuno-affinity purification of targeted allergens. Art v 1.0101, Art v 2.0101, Art v 3.0201, Art v 15 A. annua 3.0202 and Art v 3.0301 were confirmed in the reference A. argyi ** A. vulgaris, while Art v 3.0101 partial sequences were A. capillaris not. Rather it appeared in Art si 3.0101, because a unique A. gmelinii ** A. lavandulifolia peptide QGGEVPADCCAGVK was found. A. sieversiana 5 * A. vulgaris Quantification of pollen extracts and three components Total extracted protein per gram pollen weight from the seven Artemisa spp. ranged from the lowest 90 mg in A. gmelinii to the highest, 172 mg, in A. sieversiana (Fig. 3a). Fig. 3 Content of protein in pollen extracts (a) and three allergen Standard ELISA quantification curves were established components in extracts from seven Artemisia spp. b Differences for different allergens and isoforms (Additional file  1: Fig- between groups were analyzed by Tukey post hoc test (a) and Kruskal–Wallis test with Dunn’s test (b) *P < 0.05, **P < 0.01, ure S4), giving a range of homologous allergen content ***P < 0.001 of single allergen components in protein extracts from seven species: Art v 1 ranged from 3.4% in A. lavandu- lifolia to 7.1% in A. annua; Art v 2 from 1.0% in A. capil- Of 142 mugwort allergic patients, 39 showed negative laris to 3.6% in A. lavandulifolia, and Art v 3 from 0.3% IgE reactivity to six Chinese mugwort pollen extracts in A. sieversiana to 10.5% in A. argyi (Fig.  3b). The yield in ELISA, and in one patient only A. capillaris was rec- of natural allergens purified by mAb was approximate in ognized and in another only A. sieversiana. These 41 accordance with the result obtained by ELISA quantifica - patients had significantly lower IgE reactivity to mugwort tion (Additional file  1: Table  S3), while the productivity extract and Art v 1 and a slightly higher IgE reactivity to was significantly lower than expected because of a certain Art v 3 in ImmunoCAP. After testing with the mixture of amount of loss during the purification for highest purity. extracts spiked Art an 3 and Art ar 3, in 30 of these 41 patients there was positive IgE binding, especially with the Art v 3 positive patients (IgE reactivity to the mixture IgE binding comparison was positive in 17/19). The response in the 11 remaining Using the serum pools from the five areas in China, we patients was still negative to the mixture (Fig.  5), these compared the IgE binding of six Chinese Artemisia spp. patients were negative to Art v 1, and the IgE reactivi- with the reference extract A. vulgaris. We demonstrated ties to mugwort extract (w6 range: 0.46-5.8) and Art v 3 that the IgE binding capacity of A. annua and A. vulgaris (w233 range: 0-2.7) was low. was significantly higher than that of A. gmelinii , A. lavan- The IgE binding strength to mugwort extract of the 142 dulifolia and A. sieversiana. The IgE binding potency of individual patients, measured as ELISA OD values, was A. capillaris varied in the five areas: highest in Datong- closely related to the nArt v 1 IgE ImmunoCAP score, Shanxi and Beijing (Fig.  4a, c), and significantly lower in but not to the nArt v 3 score (Fig.  6a). However, when Shandong and Yunnan compared to A. annua, A. argyi rArt v 1.0101 and rArt v 3.0201 were coated in the ELISA and A. vulgaris (Fig.  4d, e). The IgE binding of 142 indi - assay, there was good correlation for both components vidual sera to pollen extracts from six Chinese mugwort (Fig. 6b). species again demonstrated higher IgE reactivity to A. By testing IgE reactivity of natural Art v 1, Art v 2 annua than to the other Artemisia spp., with A. lavandu- and Art v 3 homologous allergens by the ImmunoCAP lifolia and A. sieversiana the lowest (Fig. 4f ). A. annua A. argyi A. capillaris A.gmelinii A.lavandulifolia A. sieversiana Art v 1 Art v 2 Art v 3 Content of allergen components (%) Content of protein in pollen extract (%) Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 7 of 13 Datong,Shanxi Taiyuan,Shanxi a b 1.0 ** * 0.8 * ** **** * 0.6 0.4 0.2 0.0 Beijing Yantai,Shandong c d 1.0 ******** **** ** ** *** ** * 0.8 0.6 0.4 0.2 0.0 Qvjing,Yunnan e f 112/30 89/53 96/46 75/67 18/124 44/98 * *** * ** Fig. 4 IgE-reactivity of Artemisia pollen extracts with serum pools from Datong-Shanxi (a), Taiyuan-Shanxi (b), Beijing (c), Yantai-Shandong (d) and Yunnan (e) as determined by ELISA.ImmunoCAP IgE characterization of serum pools is shown in Additional file 1: Table S4. f IgE-reactivity of 142 individual sera to six Artemisia spp is shown as the OD deviation from the average of the six Artemisia spp. Black lines indicate mean OD difference. x/y numbers indicate the number of sera with higher (x) or lower OD (y) than the average of the six species A. annua A. argyi A. capillaris A. gmelinii A. lavandulifolia A. sieversiana A. vulgaris A. annua A. argyi A. capillaris A. gmelinii A. lavandulifolia A. sieversiana A. vulgaris OD 450nm OD 450nm Zhao et al. Clin Transl Allergy (2020) 10:50 Page 8 of 13 2.0 a 1000 *** nArt an 1 nArt ar 1 1.5 nArt gm 1 10 nArt si 1 1.0 0.35 0.5 0.1 0.01 0.0 0.001 0.0010.010.1 0.35 110 100 1000 Art v 1 sIgE (kUA/L) Fig. 5 IgE reactivity to six Chinese Artemisia pollen extracts and a mixture containing extracts and mAb purified nArt an 3 and nArt ar 3 nArt an 2 from serum of the 41 patients nArt ca 2 nArt si 2 ** r =0.489 s-m ** 0.35 r =0.793 s-1 r =0.149 s-3 mugwort 0.1 Art v 1 Art v 3 0.01 0.001 0.001 0.01 0.11 0.35 10 100 Art ar 2 sIgE (kUA/L) 0.01 0.11 10 100 1000 nArt an 3 nArt ar 3 CAP( KU /L) nArt ca 3 ** nArt gm 3 4 r =0.854 s-1 Art v 1.0101 nArt la 3 ** r =0.795 s-3 nArt si 3 Art v 3.0201 0.35 0.1 0.01 0.001 0.0010.010.1 0.35 110 100 1000 Art v 3 sIgE (kUA/L) 0.01 0.11 10 1001000 CAP( KU /L) Fig. 7 IgE levels to natural Art v 1 (a), Art ar 2 (b) and Art v 3 (c) homologous allergens of the same mugwort-sensitized patients Fig. 6 Correlation between ImmunoCAP scores and ELISA values. in China tested by ImmunoCAP. The cutoff value (0.35 kUA/L) was a mugwort CAP (rs-m), Art v 1 CAP (rs-1), Art v 3 CAP (rs-3) scores indicated by dashed lines with averaged ELISA scores from six Artemisia pollen extracts; b Art v 1 CAP (rs-1), Art v 3 CAP (rs-3) scores with ELISA coated with recombinant Art v 1.0101 and Art v 3.0201 A. annua A.argyi A. capillaris A. gmelinii A. lavandulifolia A.sieversiana Mixture ELISA (OD450nm) OD450nm ELISA (OD450nm) sIgE (kUA/L) sIgE (kUA/L) sIgE (kUA/L) Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 9 of 13 system, we found that the IgE positive rates and values seven pollen extracts, using serum pools from Shanxi and were quite similar for these allergens with high sequence Shandong, again a large difference was found. Inhibition identity, such as Art v 1 homologues (Fig. 7a), for Art v 2 to both allergen molecules and in two areas was highest type, Art ar 2 and Art ca 2 were slightly higher than Art with the A. argyi extract and lowest with A. sieversiana si 2 and Art an 2 (Fig. 7b), while Art v 3 homologues were compared to the other species (Additional file  1: Figure more variable, Art ca 3 was significantly lower, and the S5). In general, there was cross-reactivity in ELISA assays positive rates and IgE values of Art an 3, Art ar 3, Art gm coated with different Artemisia spp. extracts, except for 3, Art la 3 and Art si 3 were higher than Art v 3 and Art A. sieversiana (Additional file  1: Figure S6). This suggests ca 3 (Fig. 7c). that A. sieversiana pollen is not the primary sensitizing source. IgE inhibition using ImmunoCAP Mugwort ImmunoCAP assays with A. annua, A. vulgaris Discussion and A. sieversiana extracts on 16 different patients of the Here we present a comprehensive analysis of three group four groups (sensitized to Art v 1 and Art v 3 positive allergens, with amino acid sequence, quantity measure- or negative) confirmed that in the Art v 3 positive sera ment and IgE binding strength of pollen extracts, from group, the IgE inhibiting capacity was higher with the A. seven Artemisia spp. These species are representative of annua extract and lower with A. sieversiana, especially four sections of botanical classification and distribution when Art v 1 was negative (Fig.  8a, c), but this was not in China. the case in the Art v 3 negative sera group (Fig.  8b, d). The degree of allergen sequence variation in different In patient DT22 (component profile of high Art an 7 IgE Artemisa spp. is related to the phylogenic classification, and positive Art ar 2), IgE inhibition was even higher being similar if they belong to the same section, such as with A. sieversiana. These results indicate that the IgE A. vulgaris, A. argyi, and A. lavandulifolia (Fig. 2). Both binding potency was dependent on the presence of spe- Art v 1 and Art v 2 homologous allergen sequences in cific allergen molecules in the extract. Using ELISA to the seven Artemisia spp. were highly conserved, with test for inhibition to rArt v 1.0101 and rArt v 3.0201 with only a few amino acid changes, indicating a general Fig. 8 ImmunoCAP inhibition of A. annua, A. sieversiana and A. vulgaris against A. vulgaris pollen extract with four groups of Artemisia allergic patients. a patients sensitized to both Art v 1 and Art v 3; b patients sensitized to Art v 1 but not Art v 3; c patients sensitized to Art v 3 but not Art v 1; d patients not sensitized to either Art v 1 or Art v 3.X-axis is the inhibitor concentration of pollen extracts Zhao et al. Clin Transl Allergy (2020) 10:50 Page 10 of 13 cross reactivity in all species of this genus (Fig.  2a, b). IgE binding strength of the pollen extract is largely Art v 3 type is more variable: 26 new amino acid differ - dependent on the quantity of major allergens in the ences were found, mainly in A. annua and A. sieversi- extract and the sensitization profile of a patient’s serum ana. Including Art an 7 type sequences investigated in to a single component. The concentrations of pollen a previous study [17], the amino acid sequences of four extract influence the sensitivity and specificity of diagno - allergens in the seven Artemisa spp. indicated phylo- sis [31]. Here we found that IgE reactivity of six Chinese genic relationship and fit into four botanically classified Artemisia spp. measured by ELISA was mainly related to sections of this genus: Artemisia, Abrotanum, Dracun- the Art v 1 homologues content in extracts: A. argyi and culus and Absinthium. A recent report on the Art v 1 A. lavandulifolia pollen have almost identical sequences group allergen sequences from American mugwort (A. in four groups of allergens, but the content is different ludoviciana A. californica, A. frigida, and A. tridentate in Art v 1, causing significantly lower IgE binding of A. belong to Tridenta section) showed additional amino lavandulifolia. Natural pollen extract is not sufficient to acid variations, 81T and 85T, in the proline domain measure all component IgEs, especially for Art v 3 type (Fig. 2a) [29]. (Figs.  6a and 8c) where there is low content, and there Previous sequencing of cDNAs from A. vulgaris (pol- are other interfering factors, such as IgG antibodies [32]. len source assumed to be a single species) identified Moreover, for the 41 mugwort allergic patients who gave seven Art v 1 isoforms and four Art v 3 [21, 23], while we negative IgE reactivities to Chinese mugwort pollen deduced one or two variants/isoforms from each species extracts by ELISA, 30 gave positive results when coated by gene cloning and transcript assembly, verified by pro - with a mixture containing extracts spiked with mAb teomic mass spectrometry to isoform level. Mass spec- purified nArt an 3 and nArt ar 3 (Fig.  5). This indicated trometry of natural Art v 1 from A. vulgaris purified by again that the pollen extracts alone were not suitable for mAb could be matched to Art v 1.0101, while the natural in vitro IgE diagnosis, because of the low content of some allergen Art v 3 in A. vulgaris purified by mAb could be major and minor allergen molecules in pollen extracts, matched to isoallergens Art v 3.0201 and Art v 3.0301, in addition, for the patients with sensitization to minor and the first partial Art v 3.0101 peptide (37aa) to A. allergens alone, using extracts for immunotherapy may sieversiana. Since all except A. lavandulifolia are diploid not succeed or even worse [33]. [30], there are putatively two variants for each species. The commercial diagnostic from European mugwort We suspect the pollen sources used in previous research A. vulgaris was quite similar to Chinese silver mugwort, were not from a single species, A. vulgaris, but mixed A. argyi, in allergen sequence and in IgE binding potency. with A. sieversiana, commonly distributed in Europe. In Two Chinese mugwort species are worthy of attention: this study, comprehensive transcript analysis, gene spe- A. annua and A. sieversiana, both with more sequence cific cloning and identification of the allergen protein by variability than the reference A. vulgaris. The IgE bind - mass spectrometry guaranteed reliability. ing capacity of A. annua was also equivalent or slightly The first evaluation of in  vitro cross-reactivity, among higher than that of A. vulgaris, while that of A. sieversiana nine Artemisia spp., was done in the USA [1]. This was significantly lower. We consider that the IgE binding showed the inhibitory capacity of two local Artemisia capacity is determined by the quantity of the major aller- spp. (A. biennis, A. tridentate) was greater than that of gens, especially Art v 1, in given pollen extract. Sequence A. annua and A. vulgaris, and A. ludoviciana the least variations in the critical locations are very important, as potent. There was no difference in IgE binding capac - illustrated in the Amb a 1 isoforms with distinct immu- ity between E. coli-expressed recombinant Art v 1 iso- nological features [34]. In our research, the IgE values forms or Art v 3 isoforms within A. vulgaris because the were almost the same in the allergens with high sequence sequences were identical [21, 23]. From our results on identity (Fig.  7), while for Art v 3 type, the positive rates sequence diversity, we expect little difference in the Art v and IgE reactivity in the five Chinese species except A. 1 homologous isoforms, possibly with greater differences capillaris were higher than A. vulgaris: it is probable in Art v 2 and Art v 3 homologous isoforms in species that A. vulgaris was not the primary sensitizer for Chi- such as A. annua and A. sieversiana. When rArt v 1.0101 nese patients. Recombinant isoallergens with large amino and rArt v 3.0201 were used as coating antigens, ELISA acid variations from different species need to be evalu - inhibitions with different Artemisia species were not in ated in a large number of representative sera from differ - agreement with the results of Art v 1 and Art v 3 homo- ent geographic areas to get a more comprehensive view. logues quantification, but related to the sequence similar - In different geographic regions, there are different domi - ity of the coated isoform (Additional file  1: Figure S5 and nant Artemisia species with varying flowering time. Pol - Fig. 2), indicating the potential impact of isoforms on IgE len peaks and Art v 1 content levels have been reported binding. as higher during A. campestris flowering than that of A. Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 11 of 13 vulgaris [35]. Choosing the most relevant species in spe- gel and reaction to polyclonal antibodies (pAb) by Western blot; b, ELISA cific areas could improve the accuracy and efficiency of standard curve for Art v 1 allergen (mAb A7-G4-E6 and rabbit pAbs); c, ELISA standard curve for Art v 2 homologous allergen (mAb C9-C1 with diagnosis. The three allergen quantification methods rabbit pAbs); d, ELISA standard curve for Art v 3 homologous allergen established in this study could be applied in monitoring with two mAbs (mAbs A2-B8 and A9-G10) with representative different the Artemisia pollen allergen exposure and association isoforms. Figure S5..Inhibition of IgE binding to Art v 1 and Art v 3 with seven pollen extracts using two serum pools. a: inhibition ELISA coated analysis to allergy symptoms. with rArt v 1.0101, serum pool from Datong, Shanxi (nArt v 1: 11 kUA/l); b: inhibition ELISA coated with rArt v 1.0101, serum pool from Yantai, Shan- Conclusions dong (nArt v 1: 6.51 kUA/l); c: inhibition ELISA coated with rArt v 3.0201, serum pool from Datong, Shanxi (CAP nArt v 3: 11.27 kUA/l); d: inhibition The commercial European mugwort ImmunoCAP (A. ELISA coated with rArt v 3.0201, serum pool from Yantai, Shandong (CAP vulgaris) extract has entered the Chinese diagnostics nArt v 3: 9.65 kUA/l). Figure S6. Inhibition of sera pool from Shanxi with market, and this research indicates its general suitability extract from different species at 100 μg/ml in ELISA coated with 10 μg/ml of different pollen extracts and mixture. 6-mix, mixture of six Artemisia spp. in China as in vitro test. Our study demonstrated that A. extract in the same proportions. sieversiana, the current laboratory-based mugwort pol- len extract used for diagnosis in China, is not sufficient Abbreviation due to the low concentration of major allergen Art v 3 IUIS: International Union of Immunological Societies; mAb: Monoclonal anti- type in extract, especially for those patients who are sen- body; pAb: Polyclonal antibody; ELISA: Enzyme-linked immunosorbent assay. sitized to Art v 3 homologous allergens. A. annua and Acknowledgements A. argyi pollens are potentially suitable sources for both We thank professor Richard E. Goodman, Chairman of the Executive Commit- diagnosis and immunotherapy, the former extract has tee of WHO/IUIS Allergen Nomenclature Sub-committee for evaluating and been chosen as a sublingual immunotherapy product including new allergens reported in this paper. Dr. Ma Yintao participated in the sequencing of putative allergen genes with genomic DNAs. The authors for seasonal allergic rhinitis [36]. There is high sequence thank Lingying Wu, Yifei Wang and Xiaojing Du for their assistance in sample identity of the major mugwort allergens in seven differ - collection and testing. ent mugwort species which are common in China. Dif- Authors’ contributions ferences in IgE binding capacities among pollen extracts ZSG, WYF, RVR, FF, GG, RV, ZC conceived and designed the study. LZ, WYF, BYG, from the seven mugwort species were mainly due to vari- YL and SAV purified and characterized new allergen components. HYW, XQZ, ations in the quantity of major allergens. We therefore YF, MLL, YMS, XYW, HTW, TFL, DYW, XFW and FML were involved in the clinical study of patients. LZ, WYF, YL, HTW, TFL, BYG, SDW and YMS conducted Immu- consider that purified mugwort pollen allergen compo - noCAP testing and data analysis. LZ, WYF, ZSG, RvR, GG, SV, RV, JHA drafted the nents from A. annua and A. argyi are better suited for manuscript in collaboration with all co-authors. All authors read and approved diagnosis and treatment than crude pollen extracts which the final manuscript. have considerable variations in IgE binding capacity and Funding major allergen content. This study was carried out with financial support from Hangzhou Aileji Biotech Company, Hangzhou, China (Contract 2016R1), China-Austria Scientific Cooperation project (2019YFE0106600 and CN 14/2020), the National Natural Supplementary information Science Foundation of China (31772271) and Beijing Municipal Administra- Supplementary information accompanies this paper at https ://doi. tion of Hospitals Clinical Medicine Development of Special Funding Support org/10.1186/s1360 1-020-00354 -7. (ZYLX201826). Availability of data and materials Additional file 1: Table S1. Clinical and demographic data of 150 The new allergens reported in this manuscript, shown in Additional file 1: mugwort pollen-allergic individuals sIgE against mugwort extract (w6), Table S2, were deposited in GenBank with accession numbers. Art v 1 (w231) and Art v 3(w233) determined by ImmunoCAP, ND, not determined. AS asthma; AR allergic rhinitis; C, conjunctivitis; E, eczema. Ethics approval and consent to participate I-1, I-2, I-3, I-4 indicates the patients serum used in ImmunoCAP inhibi- Written consent was obtained from all participants (or their representatives) tion assay belonging to four groups of different sensitization patterns (1, and the study was approved by the local ethics committee (authorization Art v 1 and Art v 3 positive; 2, Art v 1 positive, Art v 3 negative; 3, Art v 1 No. 2011-R-1 and 2019-312, Second Affiliated Hospital, College of Medicine, negative, Art v 3 positive; 4, Art v 1 and Art v 3 negative). The 82 patients Zhejiang University), in collaboration with the Third People’s Hospital of 17, 20, 28 reported in previously studies are indicated by an asterisk. Table S2. Datong, Shanxi (authorization No. 2015-001), the First Affiliated Hospital, GenBank accession numbers for three allergen groups in seven Artemisia Shanxi Medical University (2019 K-K0007), Beijing Shijitan Hospital, Capital species.Table S3. Productivity of the three group allergens purified Medical University (authorization No. 2015-003). Yu Huang Ding Hospital, by specific mAb. Table S4. ImmunoCAP IgE characterization of serum Yantai, Shandong (authorization No. 2015-164), and Qvjing Chinese Traditional pools from five areas. Figure S1. Six Artemisia species collected from Medicine Hospital, Yunnan, China. The antibodies were produced by Hua An China. Figure S2. SDS-PAGE of natural purified Art v 1, Art v 2 and Art v 3 Biotech Ltd., Hangzhou, China (authorization No. SCXK 2016-0004 and SCXK homologous allergens from six Chinese Artemisia species. a, natural Art v 1 2017-0004). homologues purified by specific mAb A7-G4-E6; b, natural Art v 2 homo - logues purified by specific mAb C9-C1 shown in six different gels; c, natu- Competing interest ral Art v 3 homologues purified by specific mAb A2-B8. Figure S3. Mass Gao ZS received a grant from Hangzhou Aileji Biotech Company, Hangzhou, spectra of natural purified Art v 1(a), Art v 2(b) and Art v 3(c) homologues. China (Contract 2016R1). Gao ZS, Liu Y and Feng Y received funds from The The peptides verified by LC–MS/MS are shown in red and highlighted. Ministry of Science and Technology China (2019YFE0106600). Wang XY Figure S4. ELISA quantification of three allergen components in Artemisia received a grant from Beijing Municipal Administration of Hospitals Clinical spp. pollen. a, Chinese silver mugwort (A. argyi) pollen extract (ArE) in SDS Medicine Development of Special Funding Support (ZYLX201826). Rudolf Zhao et al. Clin Transl Allergy (2020) 10:50 Page 12 of 13 Valenta has received research grants from Viravaxx, Vienna, Austria and HVD artemisiifolia in northern China. J Zhejiang University Science B. Life Sciences, Vienna, Austria and serves as a consultant for Viravaxx. Susanne 2013;14(3):240–6. Vrtala received funds from the Austrian government (CN 14/2020). A patent 13. Li XY, Xiao XJ, Sun HZ, He SH, Yang PC, Liu ZG. Identification and for Artemisia pollen allergen nsLTP quantification by ELISA has been applied purification of major allergens in Artemisia sieversiana pollen. Chinese J in China (202011210352.1, pending) by ZS Gao, L Zhao, BY Gao and WY Fu. 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Correlation of serum allergy (IgE) 2020;75:2026–36. tests performed by different assay systems. J Allergy Clin Immunol. 2008;121:1219–24. Publisher’s Note 35. Grewling L, Bogawski P, Kostecki L, Nowak M, Szymanska A, Fratczak A. Springer Nature remains neutral with regard to jurisdictional claims in pub- Atmospheric exposure to the major Artemisia pollen allergen (Art v 1): lished maps and institutional affiliations. Seasonality, impact of weather, and clinical implications. Sci Total Environ. 2020;713:136611. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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Abstract

Background: Artemisia weed pollen allergy is important in the northern hemisphere. While over 350 species of this genus have been recorded, there has been no full investigation into whether different species may affect the allergen diagnosis and treatment. This study aimed to evaluate the variations in amino acid sequences and the content of major allergens, and how these affect specific IgE binding capacity in representative Artemisia species. Methods: Six representative Artemisia species from China and Artemisia vulgaris from Europe were used to deter- mine allergen amino acid sequences by transcriptome, gene sequencing and mass spectrometry of the purified allergen component proteins. Sandwich ELISAs were developed and applied for Art v 1, Art v 2 and Art v 3 allergen quantification in different species. Aqueous pollen extracts and purified allergen components were used to assess IgE binding by ELISA and ImmunoCAP with mugwort allergic patient serum pools and individual sera from five areas in China. Results: The Art v 1 and Art v 2 homologous allergen sequences in the seven Artemisia species were highly con- served. Art v 3 type allergens in A. annua and A. sieversiana were more divergent compared to A. argyi and A. vulgaris. The allergen content of Art v 1 group in the seven extracts ranged from 3.4% to 7.1%, that of Art v 2 from 1.0% to 3.6%, and Art v 3 from 0.3% to 10.5%. The highest IgE binding potency for most Chinese Artemisia allergy patients was with A. annua pollen extract, followed by A. vulgaris and A. argyi, with A. sieversiana significantly lower. Natural Art v 1-3 isoallergens from different species have almost equivalent IgE binding capacity in Artemisia allergic patients from China. Conclusion and clinical relevance: There was high sequence similarity but different content of the three group allergens from different Artemisia species. Choice of Artemisia annua and A. argyi pollen source for diagnosis and immunotherapy is recommended in China. Keywords: Pollen allergen, Artemisia, Different species, IgE binding potency, Allergen quantification Background Artemisia species are wind-pollinated weeds, widely dis- tributed in the northern hemisphere with a few species *Correspondence: gaozhongshan@zju.edu.cn † in the southern hemisphere [1]. Pollens of Artemisia have Lan Zhao and Wanyi Fu should be considered joint first author Allergy Research Center, Zhejiang University, Hangzhou 310058, China been recognized as a major cause of late summer and Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhao et al. Clin Transl Allergy (2020) 10:50 Page 2 of 13 autumn seasonal allergic respiratory disease, especially be better for immunotherapy (Bai et  al. China Patent, along the Asia-Europe silk-road and in north-western CN102512673B). With a serum pool from the USA, high United States [1–6]. Between 350 and 500 Artemisia levels of cross-reactivity has been found with ELISA species have been recorded in the plant kingdom [1, 7] inhibition in nine Artemisia species, with two local sage worldwide, 187 in China [8]. Phylogeny of the Artemisia species being the strongest inhibitors [1]. Very recently, genus, updated by molecular marker analysis [7, 9], has using immunoblots, similar IgE binding patterns of seven reached a consensus of six sections: Artemisia, Abro- Artemisia species have been found, with some degree of tanum, Dracunculus, Absinthium, Seriphidium and difference in three major allergen bands [17]. Tridentata. Most Artemisia species are in the first four Cross-reactivity has been found in different Artemisia sections and are distributed in temperate climate regions, species [1], but whether different species in China have where the majority of mugwort pollen allergic patients an impact on the allergen diagnosis and treatment has live. The few species belonging to Seriphidium and Tri - not been fully investigated. This study aims to provide a dentata are distributed in semi-desertic to steppic envi- comprehensive analysis of sequence variation of different ronments [10]. Some Artemisia species are dominant in isoforms and variants, content of allergens Art v 1, Art v natural plantations, contributing to the geographic dif- 2 and Art v 3, and their impact on IgE binding of six rep- ference of the pollen allergy [5]. Artemisia vulgaris is the resentative Artemisia species in China. best studied species, mainly distributed in northwestern and central Europe. Five major species have been listed Materials and methods in China (A. annua, argyi, sieversiana, capillaris, lavan- A graphic research design is presented in Fig.  1, with dulifolia) in a national pollen survey [11], and there is detailed information given in the following sections. preliminary clinical and immunological evidence of the potential IgE binding potency of the first three species Artemisia species and protein extract [12, 13]. A few species, such as A. annua, have invaded We used pollens of seven Artemisia species: five domi - Europe and America, becoming potentially severe aller- nant in Northern China (A. annua, A. argyi, A. capillaris, genic sources [14]. Artemisia pollen allergy is directly A. sieversiana, A. gmelinii) collected from Shanxi Prov- related to the distribution of Artemisia spp., density, ince; A. lavandulifolia from Zhejiang Province in South climate [6] and risk factors [15]. Currently, commercial China collected over three years (Additional file  1: Fig- mugwort pollen allergen extract CAPs are from A. absin- ure S1), and Artemisia vulgaris pollen purchased from thium (w5) and A. vulgaris (w6), the latter being the most Allergon in Sweden. Species authenticity was verified by commonly used in diagnosis. cloning and sequencing the ITS2 on genomic DNA from Molecular characterization of Artemisia vulgaris and pollen according to a reported method [24]. Aqueous Artemisia annua has revealed seven allergens, with the protein extracts of pollen were prepared by resuspend- clinical data and reference DNA and protein sequences ing 0.2 g pollen grains in 3.5 ml PBS or 2 g in 35 ml PBS published [16, 17]. Art v 1 and Art v 3 have been shown buffer (0.14  M NaCl, 2.7  mM KCl, 7.8  mM Na HPO , 2 4 to be major allergens worldwide, and a newly identified 1.5 mM KH PO ), and shaking for 12 h at 4 °C. Extracts 2 4 group, Art an 7 also seems to be important, although were centrifuged at 10,000 g for 10  min at 4  °C, filtered its IgE values are usually much lower [3, 18–20]. By through 0.22  µm filters (Millipore), and the concentra - sequence cloning of a single species of Artemisia vulgaris tion of protein in extracts was determined using the BCA pollen, seven Art v 1 isoforms have been identified, with protein assay kit (Takara Bio, Japan). Three independent only slight variation in the C-terminal and very similar extracts from different pollen samples of each species IgE reactivity [21]. Five Art v 3 isoforms have also been from China, and one A. vulgaris sample were prepared identified, one a partial sequence by N-terminal sequenc - and used in whole protein and individual allergen quan- ing [22] and the other four by gene cloning [23]. Diver- tification. The rArt v 1.0101 and rArt v 3.0201 standards sity of group 7 allergen sequences of seven Artemisia were from previous studies [21, 23]. species has recently been reported, where two isoforms for each species have been found with over 95% identical RNA extraction and Transcriptome sequence [17]. Total RNA was extracted from pollens of six Artemisia The current commercial mugwort pollen extract used spp., collected from China, using the RNAprep pure kit for skin prick and immunotherapy in China is mainly (Tiangen, China) [17], and sequenced by BGI-Shenzhen from A. sieversiana,  even though A. annua was rec- and Hangzhou One Gene Ltd using Illumina HiSeq ognized as an important allergen source in the 1980  s 2000 (San Diego, CA, USA). De novo transcriptome [5], and a recent report states that a mixture of pollens assembly was using the Trinity software package with from three species (argyi, annua, sieversiana) would a minimum K-mer of 3 and a minimum contig size of Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 3 of 13 Fig. 1 Overview of research design and outcomes 100  bp. After extraction of allergenic protein sequences, AS-LTP-Rev: TCA TTT CAC CTT GTT GCA ATC. PCR −5 blastx was used for alignment (e-value, 1e ) between amplification with both primer pairs was using Phusion Unigenes and protein databases from the Artemisia High-Fidelity DNA Polymerase (New England Biolabs, annua genome [25]. USA). At least eight clones were selected for sequenc- ing. Nucleotide sequences and deduced amino acid Cloning of Art v 1, Art v 2 and Art v 3 homologues sequences from different pollens have been deposited in Pollen cDNA was prepared with the PrimeScript   RT GenBank. Isoallergens and variants were named follow- reagent kit with gDNA Eraser (Takara Bio, Japan) using ing the nomenclature and the updated official list of the mRNA fragments as templates. The full length of Art WHO/IUIS Allergen Nomenclature Sub-committee [26] v 1, Art v 2 and Art v 3 homologues were obtained by and have been approved. PCR using primers based on Art v 1.0101 (AF493943) sequences: Art v 1-For, 5´-AAT GGC AAA GTG TTC Natural allergen purification and protein identity ATA TGTT-3´, Art v 1 - Rev, 5´-TTA GTG AGT GGA Monoclonal antibodies (mAb) used in this study were CGG AGG AG-3´; Art v 2 sequence (AM279693) Art v from previous research [27]. A7-G4-E6 specific to Art 2-For, CCT CAT ACA GAA ACA TGG GAC, Art v 2-Rev, v 1, C9-C1 to Art v 2, and A2-B8 to Art v 3, were used TTA GTA AGG TTT CTG ACC AAC AAC; and Art v 3.02 to purify three groups of allergens from six selected (EU564845, EU454846 original sequences provided by Artemisia spp. from China as described previously [27]. Gabriele Gadermaier) Art v 3-For, 5´-ATG GCA ATG LC–MS/MS (Thermo Scientific Q Exactive) was used AAA ATGA TGA A-3´ and Art v 3- Rev, 5´-CTA GCA for identity-matching of the purified proteins to deduced TAA AGY AYT TCA C-3´for first round cloning. Accord - allergens from each species. The purity of natural aller - ing to newly assembled transcript sequences for Art v 3 gens was estimated by SDS-PAGE. group, additional cloning for A. capillaris with AC-LTP- For: ATG GCA ATR AAA ATG ATG AAGG, AC-LTP-Rev: Quantification of three components by ELISA TTC CAT GTA TTC CAG CAT AAA; and A. sieversiana A. argyi extract was used to immunize two New Zealand with AS-LTP-For: ATG GCA ATG AAA ATG ATG AAG, rabbits to produce polyclonal antibodies (pAb), injecting Zhao et al. Clin Transl Allergy (2020) 10:50 Page 4 of 13 with 500 μg protein in incomplete Freund’s adjuvant, fol- Chinese Artemisia species was assessed by ELISA, using lowed by three subcutaneous boosters of 250 μg protein sera of 142 individual mugwort-allergic patients. ELISA at intervals of 7-14  days. The quality was checked using plates (Corning, USA) were coated with 0.5 μg/well pol- both Western blot and ELISA. The antibodies were pro - len extracts in PBS buffer (pH 8.3). After blocking with duced by Hua An Biotech Ltd., Hangzhou, China. An 100  μl 5% skimmed milk, 100  μl serum pool was added, mAb (A7-G4-E6) and rabbit pAbs were used for quanti- with a negative serum pool as control. After washing, fication of Art v 1 homologous proteins. A mAb (C9-C1) 100 μl goat anti-human IgE coupled with HRP (1:3000 in and rabbit pAbs were used for quantification of Art v 2 PBS buffer) was added and bound IgE was detected using homologous proteins. A selected mAb pair (A2-B8 and TMB. The ELISA was quantified using the colorimetric biotinylated A9-G10) ELISA assay was used to quantify reaction at 450/620 nm. We also compared the IgE bind- Art v 3 homologous proteins with different recombi - ing values with rArt v 1.0101 and rArt v 3.0201 allergens nant or natural allergen standards. ELISA plates (Corn- tested by ELISA and the values of Art v 1 and Art v 3 ing, USA) were coated with 0.3  μg capture antibodies sIgE tested by ImmunoCAP. For the patients who were (A7-G4-E6, C9-C1, A2-B8) at 4 °C overnight, after block- positive in ImmunoCAP with A. vulgaris but negative in ing with 100 μL 5% skimmed milk at 37  °C for 1  h, 100 ELISA with the extracts of six Chinese Artemisia spp., μL serially diluted allergen standards and pollen extracts IgE binding capacity was further tested using a mixture were added and incubated at 37°C for 1  h. After wash- of pollen extracts of A. annua, A. argyi, and natural puri- ing, the wells were incubated with 0.3  μg biotinylated fied Art an 3 and Art ar 3 in a mass ratio of 4:4:1:1 (total detection antibodies at 37 °C for 1 h followed by incuba- of 0.5 μg/well mixture). tion with 100 μL HRP-conjugated Streptavidin (1:5000 Inhibition curves were obtained using inhibitors with dilution) at 37  °C for 1  h. Finally, 100 μL TMB (3, 3′, 5, serial dilutions of pollen extracts and recombinant A. 5′-tetramethylbenzidine) was added as colorimetric sub- vulgaris allergens in competition with a solid phase strate, and after incubation in the dark for 10  min, the coated with rArt v 1.0101 and rArt v 3.0201 for IgE bind- reaction was stopped by adding 50 μL 2 M HCl. The opti - ing, using the serum pools from Shanxi and Shandong. cal density was measured at 450/620  nm (MultSkan FC, ImmunoCAP inhibition on commercial mugwort (A. vul- Thermo Fisher, USA). For each species, the allergen con - garis) extract was with serial dilutions of pollen extracts tent was measured using three independent extracts with from three species (A. annua, A. sieversiana, A. vulgaris) duplicate wells. against individual serum from four groups of different sensitization patterns (Art v 1 and Art v 3 IgE positive or negative). Patients A total of 150 patients (Additional file  1: Table S1) allergic to mugwort were recruited from Datong-Shanxi (111); ImmunoCAP tests Taiyuan-Shanxi (11); Beijing (10); Yantai-Shandong (10), According to the sequence diversity, different natural and Qvjing-Yunnan (8) in China based on a convinc- purified allergens from the three groups were selected ing case history and positive IgE reactivity to mugwort for testing the IgE by ImmunoCAP. Allergens were bioti- extracts determined by ImmunoCAP (Thermo Fisher nylated and coupled to streptavidin‐conjugated Immu- Scientific, Uppsala, Sweden). Eighty-two of these patients noCAPs (Thermo Fisher Scientific, Uppsala, Sweden) have been reported previously [17, 20, 28]. Specific IgE to at 37 °C for 30 min and then were tested with sera of 18 the major mugwort allergen components, Art v 1 and Art individual mugwort-allergic patients. v 3 was determined by ImmunoCAP. Individual sera and serum pools from the five areas were used to assess IgE binding capacity. The sera of five non-atopic individuals Statistical analyses were pooled and used as a negative control. Written con- Data were analyzed by SPSS21.0, with a value of P < 0.05 sent was obtained from all participants (or their repre- considered significantly different. Graphs were drawn sentatives) and the study was approved by the local ethics with GraphPad Prism6.0. The ANOVA model with Tuk - committee. ey’s post hoc test was used to analyze the differences in protein content between seven Artemisia spp. Differ - ELISA binding and inhibition analyses ence in IgE reactivities was analyzed with the Friedman Pollen extracts from the seven species were used to ana- test and Dunn’s multiple comparison test. The Kruskal– lyze IgE binding by ELISA, with serum pools of patients Wallis test with Dunn’s test was used to determine the from the cities of Datong and Taiyuan in Shanxi prov- quantitative variables of the three allergen components, ince, Beijing, Yantai-Shandong and Qvjing-Yunnan. and Spearman’s correlation coefficient analysis to evalu - Further IgE reactivity of each pollen extract of the six ate correlations between ImmunoCAP scores and ELISA Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 5 of 13 values. The four-parameter dose–response curve models Table  S2). The natural allergens purified by mAb (Addi - were used to build the ELISA standard curves. tional file  1: Figure S2) were matched to the target aller- gen sequences, and no other allergens were found by Results mass spectrometry (Additional file  1: Figure S3). This Patients gave six new deduced defensin-like (Art v 1 type) pro- Among 150 patients (71 male, 79 female; age range teins in six Artemisia spp. in China, with six in the IUIS 6-62  years old, mean 30.9 ± 14.7  years), 137 (91%) were from reference A. vulgaris and the other five species diagnosed with allergic rhinitis, 43 (29%) with conjunc- (Fig.  2a). They are highly conserved at the N-terminus, tivitis, 42 (28%) with asthma and 12 (8%) with eczema. with seven variable amino acids (Fig.  2a). We identified For IgE reactivity against the major components, 103 a unique amino acid, 13  W, in the defensin-domain that (68.7%) were positive to Art v 1, 74 (49.3%) were positive was present in three of the species from China (A. annua, to Art v 3, and 61 (40.7%) were positive to both, while 24 A. capillaris, A. sieversiana) and in four American Arte- (16%) were negative to both allergens (Additional file  1: misia species [29], but not in A. vulgaris. In the proline Table S1). domain, there was 78S/P substitution in A. vulgaris, A. argyi and A. lavandulifolia, while in A. sieversiana, only Sequence variation of Art v 1, Art v 2 and Art v 3 78S was found, and in A. annua, A. capillaris and A. homologous proteins gmelinii only 78P. Three types of allergens in seven Artemisia spp. were One Art v 2 homologous isoform was obtained from identified by a joint analysis of pollen transcriptome each species, resulting in four different sequences from assembly, PCR cloning and sequencing (Additional file  1: seven species: Art v 2.0101 (MF326222), Art ar 2.0101 Fig. 2 Comparison of pollen allergen deduced amino acid sequences from different Artemisia species. a defensin-like protein (Art v 1 homologous allergen); b pathogenesis-related protein 1 (Art v 2 homologous allergen); c non-specific lipid transfer protein (Art v 3 homologous allergen). Underlined sequences indicate the allergens identified in previous research. GenBank accessions are listed in Additional file 1: Table S2 Zhao et al. Clin Transl Allergy (2020) 10:50 Page 6 of 13 and Art la 2.0101 were identical, as were Art gm 2.0101 ** and Art an 2.0101, and Art si 2.0101 had an isoform with *** *** two extra amino acids (Fig. 2b). The current reference Art ** *** A. annua * *** v 2.0101 in IUIS was deduced from AM279693, it was not A. argyi confirmed in this study. A. capillaris A. gmelinii More sequence variations were observed in the lipid A. lavandulifolia transfer proteins (Art v 3 type), with a total of nine iso- A. sieversiana forms or variants and up to 38 amino acids difference (Fig.  2c). Identical isoforms were found in different spe - cies. Two isoforms, Art an 3.0102 and Art si 3.0101, had a few specific amino acids (Fig.  2c). Most isoforms from the six Chinese Artemisa spp. were verified by mass spec - trometry after immuno-affinity purification of targeted allergens. Art v 1.0101, Art v 2.0101, Art v 3.0201, Art v 15 A. annua 3.0202 and Art v 3.0301 were confirmed in the reference A. argyi ** A. vulgaris, while Art v 3.0101 partial sequences were A. capillaris not. Rather it appeared in Art si 3.0101, because a unique A. gmelinii ** A. lavandulifolia peptide QGGEVPADCCAGVK was found. A. sieversiana 5 * A. vulgaris Quantification of pollen extracts and three components Total extracted protein per gram pollen weight from the seven Artemisa spp. ranged from the lowest 90 mg in A. gmelinii to the highest, 172 mg, in A. sieversiana (Fig. 3a). Fig. 3 Content of protein in pollen extracts (a) and three allergen Standard ELISA quantification curves were established components in extracts from seven Artemisia spp. b Differences for different allergens and isoforms (Additional file  1: Fig- between groups were analyzed by Tukey post hoc test (a) and Kruskal–Wallis test with Dunn’s test (b) *P < 0.05, **P < 0.01, ure S4), giving a range of homologous allergen content ***P < 0.001 of single allergen components in protein extracts from seven species: Art v 1 ranged from 3.4% in A. lavandu- lifolia to 7.1% in A. annua; Art v 2 from 1.0% in A. capil- Of 142 mugwort allergic patients, 39 showed negative laris to 3.6% in A. lavandulifolia, and Art v 3 from 0.3% IgE reactivity to six Chinese mugwort pollen extracts in A. sieversiana to 10.5% in A. argyi (Fig.  3b). The yield in ELISA, and in one patient only A. capillaris was rec- of natural allergens purified by mAb was approximate in ognized and in another only A. sieversiana. These 41 accordance with the result obtained by ELISA quantifica - patients had significantly lower IgE reactivity to mugwort tion (Additional file  1: Table  S3), while the productivity extract and Art v 1 and a slightly higher IgE reactivity to was significantly lower than expected because of a certain Art v 3 in ImmunoCAP. After testing with the mixture of amount of loss during the purification for highest purity. extracts spiked Art an 3 and Art ar 3, in 30 of these 41 patients there was positive IgE binding, especially with the Art v 3 positive patients (IgE reactivity to the mixture IgE binding comparison was positive in 17/19). The response in the 11 remaining Using the serum pools from the five areas in China, we patients was still negative to the mixture (Fig.  5), these compared the IgE binding of six Chinese Artemisia spp. patients were negative to Art v 1, and the IgE reactivi- with the reference extract A. vulgaris. We demonstrated ties to mugwort extract (w6 range: 0.46-5.8) and Art v 3 that the IgE binding capacity of A. annua and A. vulgaris (w233 range: 0-2.7) was low. was significantly higher than that of A. gmelinii , A. lavan- The IgE binding strength to mugwort extract of the 142 dulifolia and A. sieversiana. The IgE binding potency of individual patients, measured as ELISA OD values, was A. capillaris varied in the five areas: highest in Datong- closely related to the nArt v 1 IgE ImmunoCAP score, Shanxi and Beijing (Fig.  4a, c), and significantly lower in but not to the nArt v 3 score (Fig.  6a). However, when Shandong and Yunnan compared to A. annua, A. argyi rArt v 1.0101 and rArt v 3.0201 were coated in the ELISA and A. vulgaris (Fig.  4d, e). The IgE binding of 142 indi - assay, there was good correlation for both components vidual sera to pollen extracts from six Chinese mugwort (Fig. 6b). species again demonstrated higher IgE reactivity to A. By testing IgE reactivity of natural Art v 1, Art v 2 annua than to the other Artemisia spp., with A. lavandu- and Art v 3 homologous allergens by the ImmunoCAP lifolia and A. sieversiana the lowest (Fig. 4f ). A. annua A. argyi A. capillaris A.gmelinii A.lavandulifolia A. sieversiana Art v 1 Art v 2 Art v 3 Content of allergen components (%) Content of protein in pollen extract (%) Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 7 of 13 Datong,Shanxi Taiyuan,Shanxi a b 1.0 ** * 0.8 * ** **** * 0.6 0.4 0.2 0.0 Beijing Yantai,Shandong c d 1.0 ******** **** ** ** *** ** * 0.8 0.6 0.4 0.2 0.0 Qvjing,Yunnan e f 112/30 89/53 96/46 75/67 18/124 44/98 * *** * ** Fig. 4 IgE-reactivity of Artemisia pollen extracts with serum pools from Datong-Shanxi (a), Taiyuan-Shanxi (b), Beijing (c), Yantai-Shandong (d) and Yunnan (e) as determined by ELISA.ImmunoCAP IgE characterization of serum pools is shown in Additional file 1: Table S4. f IgE-reactivity of 142 individual sera to six Artemisia spp is shown as the OD deviation from the average of the six Artemisia spp. Black lines indicate mean OD difference. x/y numbers indicate the number of sera with higher (x) or lower OD (y) than the average of the six species A. annua A. argyi A. capillaris A. gmelinii A. lavandulifolia A. sieversiana A. vulgaris A. annua A. argyi A. capillaris A. gmelinii A. lavandulifolia A. sieversiana A. vulgaris OD 450nm OD 450nm Zhao et al. Clin Transl Allergy (2020) 10:50 Page 8 of 13 2.0 a 1000 *** nArt an 1 nArt ar 1 1.5 nArt gm 1 10 nArt si 1 1.0 0.35 0.5 0.1 0.01 0.0 0.001 0.0010.010.1 0.35 110 100 1000 Art v 1 sIgE (kUA/L) Fig. 5 IgE reactivity to six Chinese Artemisia pollen extracts and a mixture containing extracts and mAb purified nArt an 3 and nArt ar 3 nArt an 2 from serum of the 41 patients nArt ca 2 nArt si 2 ** r =0.489 s-m ** 0.35 r =0.793 s-1 r =0.149 s-3 mugwort 0.1 Art v 1 Art v 3 0.01 0.001 0.001 0.01 0.11 0.35 10 100 Art ar 2 sIgE (kUA/L) 0.01 0.11 10 100 1000 nArt an 3 nArt ar 3 CAP( KU /L) nArt ca 3 ** nArt gm 3 4 r =0.854 s-1 Art v 1.0101 nArt la 3 ** r =0.795 s-3 nArt si 3 Art v 3.0201 0.35 0.1 0.01 0.001 0.0010.010.1 0.35 110 100 1000 Art v 3 sIgE (kUA/L) 0.01 0.11 10 1001000 CAP( KU /L) Fig. 7 IgE levels to natural Art v 1 (a), Art ar 2 (b) and Art v 3 (c) homologous allergens of the same mugwort-sensitized patients Fig. 6 Correlation between ImmunoCAP scores and ELISA values. in China tested by ImmunoCAP. The cutoff value (0.35 kUA/L) was a mugwort CAP (rs-m), Art v 1 CAP (rs-1), Art v 3 CAP (rs-3) scores indicated by dashed lines with averaged ELISA scores from six Artemisia pollen extracts; b Art v 1 CAP (rs-1), Art v 3 CAP (rs-3) scores with ELISA coated with recombinant Art v 1.0101 and Art v 3.0201 A. annua A.argyi A. capillaris A. gmelinii A. lavandulifolia A.sieversiana Mixture ELISA (OD450nm) OD450nm ELISA (OD450nm) sIgE (kUA/L) sIgE (kUA/L) sIgE (kUA/L) Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 9 of 13 system, we found that the IgE positive rates and values seven pollen extracts, using serum pools from Shanxi and were quite similar for these allergens with high sequence Shandong, again a large difference was found. Inhibition identity, such as Art v 1 homologues (Fig. 7a), for Art v 2 to both allergen molecules and in two areas was highest type, Art ar 2 and Art ca 2 were slightly higher than Art with the A. argyi extract and lowest with A. sieversiana si 2 and Art an 2 (Fig. 7b), while Art v 3 homologues were compared to the other species (Additional file  1: Figure more variable, Art ca 3 was significantly lower, and the S5). In general, there was cross-reactivity in ELISA assays positive rates and IgE values of Art an 3, Art ar 3, Art gm coated with different Artemisia spp. extracts, except for 3, Art la 3 and Art si 3 were higher than Art v 3 and Art A. sieversiana (Additional file  1: Figure S6). This suggests ca 3 (Fig. 7c). that A. sieversiana pollen is not the primary sensitizing source. IgE inhibition using ImmunoCAP Mugwort ImmunoCAP assays with A. annua, A. vulgaris Discussion and A. sieversiana extracts on 16 different patients of the Here we present a comprehensive analysis of three group four groups (sensitized to Art v 1 and Art v 3 positive allergens, with amino acid sequence, quantity measure- or negative) confirmed that in the Art v 3 positive sera ment and IgE binding strength of pollen extracts, from group, the IgE inhibiting capacity was higher with the A. seven Artemisia spp. These species are representative of annua extract and lower with A. sieversiana, especially four sections of botanical classification and distribution when Art v 1 was negative (Fig.  8a, c), but this was not in China. the case in the Art v 3 negative sera group (Fig.  8b, d). The degree of allergen sequence variation in different In patient DT22 (component profile of high Art an 7 IgE Artemisa spp. is related to the phylogenic classification, and positive Art ar 2), IgE inhibition was even higher being similar if they belong to the same section, such as with A. sieversiana. These results indicate that the IgE A. vulgaris, A. argyi, and A. lavandulifolia (Fig. 2). Both binding potency was dependent on the presence of spe- Art v 1 and Art v 2 homologous allergen sequences in cific allergen molecules in the extract. Using ELISA to the seven Artemisia spp. were highly conserved, with test for inhibition to rArt v 1.0101 and rArt v 3.0201 with only a few amino acid changes, indicating a general Fig. 8 ImmunoCAP inhibition of A. annua, A. sieversiana and A. vulgaris against A. vulgaris pollen extract with four groups of Artemisia allergic patients. a patients sensitized to both Art v 1 and Art v 3; b patients sensitized to Art v 1 but not Art v 3; c patients sensitized to Art v 3 but not Art v 1; d patients not sensitized to either Art v 1 or Art v 3.X-axis is the inhibitor concentration of pollen extracts Zhao et al. Clin Transl Allergy (2020) 10:50 Page 10 of 13 cross reactivity in all species of this genus (Fig.  2a, b). IgE binding strength of the pollen extract is largely Art v 3 type is more variable: 26 new amino acid differ - dependent on the quantity of major allergens in the ences were found, mainly in A. annua and A. sieversi- extract and the sensitization profile of a patient’s serum ana. Including Art an 7 type sequences investigated in to a single component. The concentrations of pollen a previous study [17], the amino acid sequences of four extract influence the sensitivity and specificity of diagno - allergens in the seven Artemisa spp. indicated phylo- sis [31]. Here we found that IgE reactivity of six Chinese genic relationship and fit into four botanically classified Artemisia spp. measured by ELISA was mainly related to sections of this genus: Artemisia, Abrotanum, Dracun- the Art v 1 homologues content in extracts: A. argyi and culus and Absinthium. A recent report on the Art v 1 A. lavandulifolia pollen have almost identical sequences group allergen sequences from American mugwort (A. in four groups of allergens, but the content is different ludoviciana A. californica, A. frigida, and A. tridentate in Art v 1, causing significantly lower IgE binding of A. belong to Tridenta section) showed additional amino lavandulifolia. Natural pollen extract is not sufficient to acid variations, 81T and 85T, in the proline domain measure all component IgEs, especially for Art v 3 type (Fig. 2a) [29]. (Figs.  6a and 8c) where there is low content, and there Previous sequencing of cDNAs from A. vulgaris (pol- are other interfering factors, such as IgG antibodies [32]. len source assumed to be a single species) identified Moreover, for the 41 mugwort allergic patients who gave seven Art v 1 isoforms and four Art v 3 [21, 23], while we negative IgE reactivities to Chinese mugwort pollen deduced one or two variants/isoforms from each species extracts by ELISA, 30 gave positive results when coated by gene cloning and transcript assembly, verified by pro - with a mixture containing extracts spiked with mAb teomic mass spectrometry to isoform level. Mass spec- purified nArt an 3 and nArt ar 3 (Fig.  5). This indicated trometry of natural Art v 1 from A. vulgaris purified by again that the pollen extracts alone were not suitable for mAb could be matched to Art v 1.0101, while the natural in vitro IgE diagnosis, because of the low content of some allergen Art v 3 in A. vulgaris purified by mAb could be major and minor allergen molecules in pollen extracts, matched to isoallergens Art v 3.0201 and Art v 3.0301, in addition, for the patients with sensitization to minor and the first partial Art v 3.0101 peptide (37aa) to A. allergens alone, using extracts for immunotherapy may sieversiana. Since all except A. lavandulifolia are diploid not succeed or even worse [33]. [30], there are putatively two variants for each species. The commercial diagnostic from European mugwort We suspect the pollen sources used in previous research A. vulgaris was quite similar to Chinese silver mugwort, were not from a single species, A. vulgaris, but mixed A. argyi, in allergen sequence and in IgE binding potency. with A. sieversiana, commonly distributed in Europe. In Two Chinese mugwort species are worthy of attention: this study, comprehensive transcript analysis, gene spe- A. annua and A. sieversiana, both with more sequence cific cloning and identification of the allergen protein by variability than the reference A. vulgaris. The IgE bind - mass spectrometry guaranteed reliability. ing capacity of A. annua was also equivalent or slightly The first evaluation of in  vitro cross-reactivity, among higher than that of A. vulgaris, while that of A. sieversiana nine Artemisia spp., was done in the USA [1]. This was significantly lower. We consider that the IgE binding showed the inhibitory capacity of two local Artemisia capacity is determined by the quantity of the major aller- spp. (A. biennis, A. tridentate) was greater than that of gens, especially Art v 1, in given pollen extract. Sequence A. annua and A. vulgaris, and A. ludoviciana the least variations in the critical locations are very important, as potent. There was no difference in IgE binding capac - illustrated in the Amb a 1 isoforms with distinct immu- ity between E. coli-expressed recombinant Art v 1 iso- nological features [34]. In our research, the IgE values forms or Art v 3 isoforms within A. vulgaris because the were almost the same in the allergens with high sequence sequences were identical [21, 23]. From our results on identity (Fig.  7), while for Art v 3 type, the positive rates sequence diversity, we expect little difference in the Art v and IgE reactivity in the five Chinese species except A. 1 homologous isoforms, possibly with greater differences capillaris were higher than A. vulgaris: it is probable in Art v 2 and Art v 3 homologous isoforms in species that A. vulgaris was not the primary sensitizer for Chi- such as A. annua and A. sieversiana. When rArt v 1.0101 nese patients. Recombinant isoallergens with large amino and rArt v 3.0201 were used as coating antigens, ELISA acid variations from different species need to be evalu - inhibitions with different Artemisia species were not in ated in a large number of representative sera from differ - agreement with the results of Art v 1 and Art v 3 homo- ent geographic areas to get a more comprehensive view. logues quantification, but related to the sequence similar - In different geographic regions, there are different domi - ity of the coated isoform (Additional file  1: Figure S5 and nant Artemisia species with varying flowering time. Pol - Fig. 2), indicating the potential impact of isoforms on IgE len peaks and Art v 1 content levels have been reported binding. as higher during A. campestris flowering than that of A. Zhao  et al. Clin Transl Allergy (2020) 10:50 Page 11 of 13 vulgaris [35]. Choosing the most relevant species in spe- gel and reaction to polyclonal antibodies (pAb) by Western blot; b, ELISA cific areas could improve the accuracy and efficiency of standard curve for Art v 1 allergen (mAb A7-G4-E6 and rabbit pAbs); c, ELISA standard curve for Art v 2 homologous allergen (mAb C9-C1 with diagnosis. The three allergen quantification methods rabbit pAbs); d, ELISA standard curve for Art v 3 homologous allergen established in this study could be applied in monitoring with two mAbs (mAbs A2-B8 and A9-G10) with representative different the Artemisia pollen allergen exposure and association isoforms. Figure S5..Inhibition of IgE binding to Art v 1 and Art v 3 with seven pollen extracts using two serum pools. a: inhibition ELISA coated analysis to allergy symptoms. with rArt v 1.0101, serum pool from Datong, Shanxi (nArt v 1: 11 kUA/l); b: inhibition ELISA coated with rArt v 1.0101, serum pool from Yantai, Shan- Conclusions dong (nArt v 1: 6.51 kUA/l); c: inhibition ELISA coated with rArt v 3.0201, serum pool from Datong, Shanxi (CAP nArt v 3: 11.27 kUA/l); d: inhibition The commercial European mugwort ImmunoCAP (A. ELISA coated with rArt v 3.0201, serum pool from Yantai, Shandong (CAP vulgaris) extract has entered the Chinese diagnostics nArt v 3: 9.65 kUA/l). Figure S6. Inhibition of sera pool from Shanxi with market, and this research indicates its general suitability extract from different species at 100 μg/ml in ELISA coated with 10 μg/ml of different pollen extracts and mixture. 6-mix, mixture of six Artemisia spp. in China as in vitro test. Our study demonstrated that A. extract in the same proportions. sieversiana, the current laboratory-based mugwort pol- len extract used for diagnosis in China, is not sufficient Abbreviation due to the low concentration of major allergen Art v 3 IUIS: International Union of Immunological Societies; mAb: Monoclonal anti- type in extract, especially for those patients who are sen- body; pAb: Polyclonal antibody; ELISA: Enzyme-linked immunosorbent assay. sitized to Art v 3 homologous allergens. A. annua and Acknowledgements A. argyi pollens are potentially suitable sources for both We thank professor Richard E. Goodman, Chairman of the Executive Commit- diagnosis and immunotherapy, the former extract has tee of WHO/IUIS Allergen Nomenclature Sub-committee for evaluating and been chosen as a sublingual immunotherapy product including new allergens reported in this paper. Dr. Ma Yintao participated in the sequencing of putative allergen genes with genomic DNAs. The authors for seasonal allergic rhinitis [36]. There is high sequence thank Lingying Wu, Yifei Wang and Xiaojing Du for their assistance in sample identity of the major mugwort allergens in seven differ - collection and testing. ent mugwort species which are common in China. Dif- Authors’ contributions ferences in IgE binding capacities among pollen extracts ZSG, WYF, RVR, FF, GG, RV, ZC conceived and designed the study. LZ, WYF, BYG, from the seven mugwort species were mainly due to vari- YL and SAV purified and characterized new allergen components. HYW, XQZ, ations in the quantity of major allergens. We therefore YF, MLL, YMS, XYW, HTW, TFL, DYW, XFW and FML were involved in the clinical study of patients. LZ, WYF, YL, HTW, TFL, BYG, SDW and YMS conducted Immu- consider that purified mugwort pollen allergen compo - noCAP testing and data analysis. LZ, WYF, ZSG, RvR, GG, SV, RV, JHA drafted the nents from A. annua and A. argyi are better suited for manuscript in collaboration with all co-authors. All authors read and approved diagnosis and treatment than crude pollen extracts which the final manuscript. have considerable variations in IgE binding capacity and Funding major allergen content. This study was carried out with financial support from Hangzhou Aileji Biotech Company, Hangzhou, China (Contract 2016R1), China-Austria Scientific Cooperation project (2019YFE0106600 and CN 14/2020), the National Natural Supplementary information Science Foundation of China (31772271) and Beijing Municipal Administra- Supplementary information accompanies this paper at https ://doi. tion of Hospitals Clinical Medicine Development of Special Funding Support org/10.1186/s1360 1-020-00354 -7. (ZYLX201826). Availability of data and materials Additional file 1: Table S1. Clinical and demographic data of 150 The new allergens reported in this manuscript, shown in Additional file 1: mugwort pollen-allergic individuals sIgE against mugwort extract (w6), Table S2, were deposited in GenBank with accession numbers. Art v 1 (w231) and Art v 3(w233) determined by ImmunoCAP, ND, not determined. AS asthma; AR allergic rhinitis; C, conjunctivitis; E, eczema. Ethics approval and consent to participate I-1, I-2, I-3, I-4 indicates the patients serum used in ImmunoCAP inhibi- Written consent was obtained from all participants (or their representatives) tion assay belonging to four groups of different sensitization patterns (1, and the study was approved by the local ethics committee (authorization Art v 1 and Art v 3 positive; 2, Art v 1 positive, Art v 3 negative; 3, Art v 1 No. 2011-R-1 and 2019-312, Second Affiliated Hospital, College of Medicine, negative, Art v 3 positive; 4, Art v 1 and Art v 3 negative). The 82 patients Zhejiang University), in collaboration with the Third People’s Hospital of 17, 20, 28 reported in previously studies are indicated by an asterisk. Table S2. Datong, Shanxi (authorization No. 2015-001), the First Affiliated Hospital, GenBank accession numbers for three allergen groups in seven Artemisia Shanxi Medical University (2019 K-K0007), Beijing Shijitan Hospital, Capital species.Table S3. Productivity of the three group allergens purified Medical University (authorization No. 2015-003). Yu Huang Ding Hospital, by specific mAb. Table S4. ImmunoCAP IgE characterization of serum Yantai, Shandong (authorization No. 2015-164), and Qvjing Chinese Traditional pools from five areas. Figure S1. Six Artemisia species collected from Medicine Hospital, Yunnan, China. The antibodies were produced by Hua An China. Figure S2. SDS-PAGE of natural purified Art v 1, Art v 2 and Art v 3 Biotech Ltd., Hangzhou, China (authorization No. SCXK 2016-0004 and SCXK homologous allergens from six Chinese Artemisia species. a, natural Art v 1 2017-0004). homologues purified by specific mAb A7-G4-E6; b, natural Art v 2 homo - logues purified by specific mAb C9-C1 shown in six different gels; c, natu- Competing interest ral Art v 3 homologues purified by specific mAb A2-B8. Figure S3. Mass Gao ZS received a grant from Hangzhou Aileji Biotech Company, Hangzhou, spectra of natural purified Art v 1(a), Art v 2(b) and Art v 3(c) homologues. China (Contract 2016R1). Gao ZS, Liu Y and Feng Y received funds from The The peptides verified by LC–MS/MS are shown in red and highlighted. Ministry of Science and Technology China (2019YFE0106600). Wang XY Figure S4. ELISA quantification of three allergen components in Artemisia received a grant from Beijing Municipal Administration of Hospitals Clinical spp. pollen. a, Chinese silver mugwort (A. argyi) pollen extract (ArE) in SDS Medicine Development of Special Funding Support (ZYLX201826). Rudolf Zhao et al. Clin Transl Allergy (2020) 10:50 Page 12 of 13 Valenta has received research grants from Viravaxx, Vienna, Austria and HVD artemisiifolia in northern China. J Zhejiang University Science B. Life Sciences, Vienna, Austria and serves as a consultant for Viravaxx. Susanne 2013;14(3):240–6. Vrtala received funds from the Austrian government (CN 14/2020). A patent 13. Li XY, Xiao XJ, Sun HZ, He SH, Yang PC, Liu ZG. Identification and for Artemisia pollen allergen nsLTP quantification by ELISA has been applied purification of major allergens in Artemisia sieversiana pollen. Chinese J in China (202011210352.1, pending) by ZS Gao, L Zhao, BY Gao and WY Fu. 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Correlation of serum allergy (IgE) 2020;75:2026–36. tests performed by different assay systems. J Allergy Clin Immunol. 2008;121:1219–24. Publisher’s Note 35. Grewling L, Bogawski P, Kostecki L, Nowak M, Szymanska A, Fratczak A. Springer Nature remains neutral with regard to jurisdictional claims in pub- Atmospheric exposure to the major Artemisia pollen allergen (Art v 1): lished maps and institutional affiliations. Seasonality, impact of weather, and clinical implications. Sci Total Environ. 2020;713:136611. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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