s2fieldsofstudy: Biology Medicine

text:
TNF-α G-308A genetic variants, serum CRP-hs concentration and DNA damage in obese women

Obesity is associated with inflammation, which can disturb genome stability. Tumor necrosis factor (TNF-α) polymorphism was found to affect TNF-α protein production and inflammation. Therefore, the present study illustrates the relationship between TNF-α polymorphism, the degree of inflammation assessed by serum high sensitivity C-reactive protein concentration (CRP-hs) and basal DNA damage in patients with obesity (BMI 30–34.9 kg/m2) and control subjects with proper body mass (BMI < 25 kg/m2). A total of 115 participants (75 obese premenopausal women; and 40 age-, and gender-matched controls) were included. Biochemical parameters (serum concentrations of total-cholesterol, HDL-cholesterol, LDL- cholesterol, triglycerides, glucose, apolipoprotein AI, CRP-hs) and endogenous DNA damage (determined by comet assay) were measured. TNF-α G-308A polymorphism (rs1800629) was analyzed by PCR-RFLP (PCR-restriction fragments length polymorphism). An effect of TNF-α genotype on serum CRP-hs concentration was noted (p = 0.031). In general, carriers of the rare A allele of the TNF-α G-308A polymorphism had significantly lower endogenous DNA damage and serum CRP-hs concentrations than GG homozygotes, however, the protective effect of the A allele was especially visible in non-obese women. Serum CRP-hs concentrations and levels of DNA damage (% DNA in tail) were significantly higher in obese than in controls (p = 0.001 and p < 0.0001, respectively). The adjusted multiple linear regression analyses revealed a significant, independent impact of obesity on DNA damage (p = 0.00000) and no effect of other covariates i.e. age, TNF-α genotype and serum CRP-hs concentration. Our study showed that obesity has a significant impact on the levels of endogenous DNA damage. Obesity abolished the protective effect of A allele of the TNF-α G-308A polymorphism on DNA damage and on inflammation development observed in non-obese A allele carriers.

Introduction
Smoking, improper diet and environmental toxins have been recognized as main exogenous sources of DNA damage [1]. However, besides exogenous factor-caused DNA breaks, endogenous DNA damage and failure of DNA repair can disturb cell metabolism and function [2]. In cells, production of reactive oxygen species (ROS) and inflammation have been recognized to cause DNA lesions [3]. DNA damage has been found to be involved in aging and development of common diseases including cancer, atherosclerosis, metabolic syndrome [4][5][6].
The Comet Assay is a sensitive and low-cost technique, which measures DNA damage in individual cells [7,8]. In addition to DNA strand breaks (double strand breaks and single strand breaks), the modified Comet Assay serve to detect also oxidized bases, interstrand cross-links or The original version of this article was revised. misincorporated uracil [9]. This method is widely used to determine the level of DNA damage, both resulting from exposure to environmental mutagens, as well as arising in the course of many diseases [10][11][12].
Obesity is a worldwide problem with increasing prevalence, associated with co-morbidities such as type 2 diabetes mellitus and cardiovascular diseases, and increased cancer risk [13][14][15]. Obesity is characterized by the, adipocyte hypertrophy, elevated production of reactive oxygen species, cytokines, chronic inflammation, disturbances in insulin and glucose metabolism [16][17][18]. Association between the occurrence of DNA lesions and enhanced body weight has been also reported [19][20][21]. Both inflammation and metabolic disturbances can cause DNA damage [22][23][24]. Moreover, the relationship between chronic inflammation and genomic instability has been observed in about 25% of human cancers [25][26][27].
Tumor necrosis factor-alpha (TNF-α) is a multi-functional cytokine synthesized by adipocytes, preadipocytes, endothelial cells, smooth muscle cells, fibroblasts, leukocytes and macrophages [28][29][30][31]. It can participate in regulation of many cellular processes such as immune function, differentiation, proliferation, apoptosis and energy pathways [32,33]. Variations in the TNF-α gene can affect TNF-α production and a significant effect of the polymorphism in the promoter region of the TNF-α gene at position -308 (rs1800629) was reported [34][35][36]. Presence of the variant allele has been shown to increase the rate of transcription and production of the TNF-α protein [37][38][39]. G-308A polymorphism in the TNF-α gene has been associated with the development of inflammation and risk of cardiovascular diseases [40][41][42][43][44]. A recent study revealed the predisposition of GG homozygotes to higher production of pro-inflammatory molecules resulting in their enhanced serum levels [45]. The G-308A polymorphism was also found to be associated with insulin sensitivity and increased production of leptin, suggesting an impact of TNF-α gene on obesity and obesityrelated health complications [46]. Phillips et al. showed that patients carrying the GG genotype had elevated risk of metabolic syndrome compared with carriers of the minor A allele [47]. However, large cohort studies in Chinese, Caucasians and Afro-Americans did not show a significant correlation between G-308A polymorphism and insulin resistance or obesity [48][49][50][51]. Recently published systematic review and meta-analysis have indicated an association between TNF-α G-308A gene polymorphism and the risk of ischemic heart disease [28,52].
TNF-α stimulates the production of C-reactive protein (CRP) and the development of inflammatory processes, and serum CRP concentration is commonly used as a marker of inflammation [53][54][55][56]. Furthermore, in vitro studies showed increased DNA damage as a result of TNF-α stimulated ROS production [57]. TNF-α together with IL-1β and IFN-γ induced DNA damage in human cholangiocarcinoma cell line [58]. DNA damage and enhanced ROS levels were related to TNF-α-mediated senescence in HUVEC (human umbilical vein endothelial cells) [59].
The aim of our study was to assess the impact of the G-308A TNF-α polymorphism on CRP-hs levels and genomic stability measured by basal DNA damage in obese women.

Subjects
The study participants were premenopausal women (Polish Caucasians from the Warsaw region). Among 115 participants of the study, 75 were obese. Obesity was classified according to World Health Organization criteria [60] i.e., subjects with BMI ≥ 30 kg/m 2 were considered obese. The obese women were consecutively recruited between December 2011 and June 2013 on the basis of clinical assessments from subjects who had been directed to the Outpatient Clinic at the National Food and Nutrition Institute in Warsaw due to obesity treatment. The gender-and age-matched control group (n = 40) of apparently healthy women with proper body mass (BMI not exceeded 25 kg/m 2 ) was recruited from subjects directed for a routine general health screening. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and the Local Ethics Committee at the National Food and Nutrition Institute approved all procedures involving human subjects. Written informed consent was obtained from all of the registered volunteers.
The recruited women were premenopausal, non-smoking (for at least 5 years), had no history of alcoholism, and had no signs or symptoms of renal and hepatic disorders, endocrine disorders (e.g. disease of the thyroid, parathyroid, Cushing's syndrome, polycystic ovary syndrome), autoimmune diseases, and cancer. Women within the last 3 months before the study were not receiving medications known to influence plasma lipid levels and did not use hormonal therapy as well as did not report chronic use of dietary supplements and anti-inflammatory drugs. Exclusion factors were also menopause, pregnancy, and lactation.

Anthropometric measurements
All subjects underwent a comprehensive medical evaluation including medical history, physical examination and measurement of anthropometric parameters: body weight, body height, waist circumference, hip circumference according to standardized procedures routinely performed in the Outpatient Clinic at the National Food and Nutrition Institute (Warsaw, Poland). The body waist circumference was 1 3 measured at the midpoint between the lower margin of the last rib cage and the top iliac crest by using a flexible inch tape. Measurements were taken in the morning, after an overnight fasting, at the same day, or the day before blood samplings. Based on anthropometric measurements the BMI and WHR (waist-hip ratio) indexes were calculated.

Blood analysis
Blood was collected after night fasting from all subjects and serum parameters were analyzed on the same day. Total cholesterol, HDL-cholesterol, triglycerides, glucose, and insulin were measured using standard techniques in a certified laboratory for clinical chemistry at The National Food and Nutrition Institute. The LDL cholesterol levels were calculated using the Friedewald formula. Residue serum was aliquoted and frozen at − 20 °C until analysis. CRP-hs concentrations were obtained using commercially available ELISA (Immundiagnostik AG, Germany), according to the protocol provided by the manufacturer. The serum concentrations of apolipoprotein AI were measured using monoclonal antibodies against apolipoprotein AI (Pointe Scientific, USA) by the immunotubidymetric method.

Genotype analysis
Genomic DNA was extracted from peripheral white blood cells of whole-blood samples using DNA Mini Kit (A&A Biotechnology, Poland). Extracted DNA samples (100 ng) were amplified to obtain a fragment including the polymorphic region of TNF-α G-308A gene (rs 1,800,629), as previously described [61] by using the following primers: F5′-AAT AGG TTT TGA GGG CCA TG-3′ and R5′-GGG ACA CAC AAG CAT CAA GG-3′. Approximately 100 ng of DNA was amplified by thermal cycling using the DNA polymerase kit (BioLine, London, UK) in 25 µL of PCR mixture containing 2.5 mM MgCl 2 , 0.4 mM of each deoxyribonucleotide triphosphate (dNTP, New England Biolabs, USA), 1 U Taq, and 100 pM of each primer. Polymerase chain reaction conditions included an initial denaturation at 94 °C for 5 min followed by 35 cycles of 94 °C for 15 s, 55 °C for 30 min, and 72 °C for 15 s, with a final extension at 72 °C for 7 min. The amplified DNA samples containing a polymorphic site was digested with the restriction enzyme NcoI (New England Biolabs, USA) and products were run on agarose gel electrophoresis. Digestion of the 151 bp fragment carrying the G allele was giving 139 bp and 12 bp fragments, while the fragment with the A allele remained intact. About 20% of all samples were randomly selected for repeated genotyping for confirmation. Concordance between repeats was 100%.

Comet assay
DNA integrity was determined by the use of alkaline singlecell gel electrophoresis (comet assay), based on previous reports [62 , 63]. Lymphocytes were obtained from 1 mL heparinized blood by centrifugation in a density gradient; then 50 µL of lymphocytes (1-3 × 10 5 cells/mL) was distributed with 50 µL of 2% low-melting-point agarose on a microscope slide precoated with 0.5% normal agarose. The slides were incubated for 1 h in a freshly prepared cold (4 °C) lysis solution (2.5 M NaCl, 100 mM EDTA-Na 2 , 10 mM Tris, pH 10.0-10.5) with 1% Triton X-100. Next, the slides were left in a horizontal gel electrophoresis tank with alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA-Na 2 , pH > 13.0) for 40 min at 4 °C. Electrophoresis was performed under following conditions: 20 min, 35 V (1 V/cm), 300 mA. Slides were then washed with a neutralizing solution (0.4 M Tris, pH 7.5), and stained with DAPI (20 µg/ml).Nikon Eclipse 50i fluorescence microscope (×400 magnification) and Lucia Comet Assay software version 4.81 (Laboratory Imaging, Prague, Czech Republic) was used to analyze 100 comets on each slide. From each subject three blood samples were analyzed in duplicates. Of the data obtained, % DNA in the tail was chosen for further analysis as a DNA damage parameter. The chemicals were supplied by Sigma-Aldrich.

Statistical analysis
All statistical calculations were performed with the Statistica software (version 12.0). The distribution of variables was tested by Shapiro-Wilk test. Differences in continuous parameters were tested using Mann-Whitney U-test. Spearman correlation analyses were performed for the relationships among the variables. Non-continuous variables were tested with a Chi square test. Allele frequencies for TNF-α variants were calculated with the gene counting method. Hardy-Weinberg equilibrium (HWE) was determined by Pearson's χ 2 goodness-of-fit test. CRP-hs was dichotomized as ≥ 3 mg/L versus otherwise (< 3 mg/L) based on the well accepted cut-off point (of 3 mg/L) indicating elevated CRPhs associated with an increased risk for CVD [64]. Regression analyses were performed using generalized linear models. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using logistic regression. Results were expressed as means ± SD or percentages, and p < 0.05 was considered statistically significant.

Results
Characteristics of the studied subjects are summarized in Table 1. Obese and control subjects (non-obese) were in similar age. There were substantial differences in serum concentrations of total cholesterol, triglycerides, LDLcholesterol, and blood pressure between studied groups (p < 0.05). While no differences in HDL-cholesterol, apolipoprotein AI and glucose concentrations were found. Serum C-reactive protein (CRP-hs) concentrations as well as mean level of DNA damage (% DNA in tail) were significantly higher in obese than in controls (Table 1).
Among all studied subjects the distribution of TNF-α gene alleles was in Hardy-Weinberg equilibrium (χ 2 value = 2.68, p = 0.100). The frequency of the rare A allele of the TNF-α gene G-308A polymorphism was 17.4%. 71% of the studied women carried the GG genotype (wildtype), 24%-the GA genotype and 5%-the AA genotype. As reported in Table 2, no statistically significant difference in frequency of the three genotypes of G-308A TNF-α polymorphism among obese and non-obese was found. However, the prevalence of obesity was higher among subjects with GG genotype than among A allele carriers (71% and 50% respectively, p = 0.026). Due to the low frequency of the AA genotype statistical analyses were performed for A allele carriers (GA and AA genotypes pooled) and GG homozygotes.
Study participants' characteristics according to the G-308A TNF-α polymorphism are presented in Table 3. In GG homozygotes higher diastolic blood pressure was observed. No statistically significant difference in mean values of BMI and WHR between analyzed groups was recognized. Carriers of the GG genotype had higher levels of DNA damage as well as higher CRP-hs serum concentrations compared to the A allele carriers (3.55 ± 1.70% vs. 2.80 ± 1.29%; p = 0.025 and 4.06 ± 2.07 mg/L vs. 2.92 ± 2.15 mg/L, respectively, p = 0.0001).
In studied group DNA damage was significantly correlated with BMI, WHR, systolic and diastolic blood pressure as well as total cholesterol, LDL cholesterol, and serum CRP-hs concentrations ( Table 4). The observed association between DNA damage and BMI was not affected by the TNF-α genotype. However, an impact of the TNF-α genotype on the associations between DNA damage and WHR and serum CRP-hs was observed. Only among A allele carriers a strong, positive correlation between DNA damage and WHR was observed (R = 0.784, p = 0.00004). Also in A allele carriers but not in GG homozygotes, a positive correlation between DNA damage and serum CRP-hs concentration was recognized (Table 4, R = 0.578, p = 0.0003).  When obese and non-obese carriers of studied genotypes were analyzed separately, obesity not TNF-α genotype was found to affect DNA damage as higher levels of DNA damage occurred in obese compared to non-obese. Obese women carrying GG genotype had only slightly more DNA damage (% DNA in tail) than obese A allele carriers (4.37 ± 1.27% and 3.84 ± 0.97%, respectively; p = 0.084). Also among nonobese women non-significant allele effect on DNA damage was observed (1.49 ± 0.43% in GG and 1.75 ± 0.41% in A allele carriers; p = 0.057). The G-308A TNF-α polymorphism was found to affect serum CRP-hs concentrations only in non-obese women. Mean serum CRP-hs concentration was significantly higher in non-obese women with GG genotype than in non-obese A allele carriers (p = 0.0001), while among obese participants similar CRP-hs levels were observed (Fig. 1). Taking into account the degree of inflammation, higher, but not significant incidence of elevated serum CRP-hs concentrations (≥ 3 mg/L) was found among obese subjects: (OR 1.97 95% CI 0.88-4.41, p = 0.095). Additionally, logistic regression analysis identified GG genotype as a risk factor for elevated CRP-hs (≥ 3 mg/L) only in non-obese women ( Table 5). The odds of CRP-hs ≥ 3 mg/L in nonobese women with GG genotype was 50 times greater than in non-obese women with A allele (p = 0.0001, OR 50.00, 95% CI 6.95-359.75). Multiple linear regression analyses revealed also a significant interaction effect of TNF-α genotype and elevated CRP-hs (≥ 3 mg/L) on the levels of DNA damage (F = 4.75, p = 0.031) in all studied women.
However, when all covariates elevated CRP-hs, TNF-α genotype, obesity and age were included into the statistical analyses, the impact of obesity (BMI ≥ 30 kg/m 2 ) on the level of DNA damage was independent of other covariates (F = 69.41, p = 0.0000) and no significant interaction effect was observed.

Discussion
Obesity, that is a result of an imbalance between energy intake and expenditure, has reached epidemic proportions with increasing prevalence worldwide. Adipose tissue participates in the production of inflammatory mediators, and in adipose tissue from obese enhanced TNF-α production was observed [51,52,[65][66][67].
The G-308A polymorphism in the promoter region of the TNF-α gene was found to affect TNF-α protein expression and ischemic heart disease risk in, both, Caucasians and Asians [68,69]. The human TNF-α protein is coded by the gene located near major histocompatibility complex (MHC) between the class I HLA-B and the class II HLA-DR loci [70,71]. Therefore, the SNPs in the TNF-α promoter may be related to HLA haplotypes and autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [72][73][74].
Low-grade chronic inflammation is a characteristic feature of obesity, and plays an important role in the pathogenesis of obesity-associated comorbidities [16]. Inflammation is linked with enhanced generation of reactive oxygen species (ROS), which can damage cellular biomolecules, including DNA, leading to disturbances in cell signaling and cell cycle control, genetic mutations, and promotion of inflammation [75].
In vitro studies recognized that pro-inflammatory cytokines provoke DNA damage, cell senescence and growth arrest [59,76,77]. In IFNγ/TNFα-induced genotoxicity, NADPH oxidases (Nox 1 and 4) and TGFβ/SMAD pathways are involved in enhanced ROS production [76]. ROS formation and increased level of DNA lesions were observed as a result of high CRP-hs in the culture of HUVECs [78].   Oxidative stress, chronic inflammation and DNA damage have been recognized as important factors leading to the development of carcinogenesis, atherosclerosis and cardiovascular diseases. Obesity is associated with elevated risk of all these diseases [79][80][81][82][83]. Therefore, not only obesityassociated inflammation but also obesity-associated DNA damage may play a significant role in the development of both cardiovascular diseases and cancer in obese [26,84]. In cancerogenesis, enhanced mutation rate was found to be linked to a high amount of DNA lesions [85,86].
The G-308A polymorphism in TNF-α gene was reported in relation to TNF-α protein production and development of inflammation as well as it was suggested to play an important role in the development and progression of cancer [87][88][89]. Therefore, this polymorphism may affect both development of inflammation and formation of DNA lesions. Thus, we hypothesized that in obese amount of endogenous DNA lesions may be linked to the degree of inflammation and TNF-α gene polymorphism, and in the present study we assessed the association between G-308A TNF-α gene variants, serum concentrations of CRP-hs and DNA damage in obese.
The present study was conducted in Caucasian women from the central region of Poland and among our study participants we found the low frequency of the AA genotype (5%) as well as A allele (17.4%) of the G-308A polymorphism in TNF-α gene. This is consistent with the results of other studies reporting that the G to A change in TNF-α gene is rather rare [90]. In a study of 120 Caucasian Italian women no AA homozygotes were recognized and the frequency of A allele was 27.4% [90]. The frequency of AA genotype was about 4% among Han Chinese [91], 2% among people with obesity from Spain [92], and 0.6% in the Brazilian individuals [93]. In GG homozygotes higher plasma levels of TNF-α and CRP-hs than in AA homozygotes [94][95][96][97] as well as in GA heterozygotes of the G-308A polymorphism in the TNF-α gene were reported [90].
In the present study non-obese carriers of the A allele had significantly lower CRP-hs serum concentrations than GG homozygotes. The presence of the A allele appears to have a protective anti-inflammatory effect, which, however, disappears when obesity appears. We observed similar CRP-hs concentrations in both obese and non-obese GG homozygotes, while obese had higher levels of DNA damage. It indicates that in obese GG homozygotes other factors than inflammation, have a significant impact on cellular DNA damage. In A allele carriers DNA damage was positively correlated with serum CRP-hs concentration and in obese-A allele carriers similar levels of serum CRP-hs and DNA damage as in obese-GG homozygotes were observed. Our study participants, both obese and controls, can be classified as apparently healthy, thus we can hypothesize that obesity and low-grade inflammation characteristic for obesity can affect basal DNA damage observed in this study. We found significantly greater amount of DNA lesions in obese than in non-obese women as well as the associations between BMI and DNA damage. It is in agreement with our previous study [21]. However, the results of the presented study show that the impact of obesity and obesity-associated disturbances on DNA damage is strong and occurrence of obesity eliminates or significantly decreases the effect of the G-308A TNF-α variants on both inflammation, and levels of DNA damage. In obesity oxidative stress and inflammation are involved in the induction of DNA lesions and have an impact on the efficiency of the DNA repair mechanisms [24]. DNA damage in cells may be induced by pro-inflammatory cytokines, chemokines and molecules such as NO (nitric oxide), and ROS [98,99]. In white adipose tissue amount of DNA damage was related to pro-inflammatory markers such as IL-6 and TNF-α [77]. As CRP-hs exerts ROS production in vitro [78] it can be hypothesized that the potential cause of DNA damage found in our study participants is oxidative stress related to enhanced inflammation (CRP-hs ≥ 3 mg/dl). Our results indicates that in non-obese, apparently healthy women GG homozygosity of the G-308A TNF-α polymorphism is associated with enhanced low grade inflammation assessed by serum CRP-hs concentrations, and occurrence of obesity does not affect significantly CRP-hs levels in GG homozygotes. The presence of A allele in non-obese women protects against inflammation but development of obesity abolished this allele effect.
A broad range of DNA lesions has been recognized in people with obesity [100][101][102]. Enhanced DNA damage was also reported in patients with obesity-related diseases such as type 2 diabetes and metabolic syndrome [103]. Moreover, body weight loss resulted in a reduction in the level of DNA damage [104][105][106][107]. The amount of DNA with oxidative damage was associated with levels of cholesterol, triglycerides and HbA1c [108]. In the present study a relationship between DNA damage and both, total cholesterol and LDLcholesterol was also observed. Our study does have some limitations and one important limitation is a small sample size, which may be responsible for the observed lower frequency of A-allele carriers among obese than among nonobese. Moreover, we studied only women, therefore, data for men as well younger and older populations containing subjects of both genders and BMI in a wide range are needed.
In summary, the present study demonstrates the strong impact of obesity on basal DNA damage assessed by the comet assay (as % DNA in tails) and indicates that the presence of obesity abolished the protective effect of A allele on inflammation development observed in non-obese women.
Regional Development Fund within the Operational Program (Innovative economy for 2007-2013).

Authors' contributions
The author's responsibilities were as follows: MW was responsible for the study concept and design, recruited patients, performed laboratory and statistical analysis, interpreted the data, and took the lead in writing the manuscript. MC created a database and contributed to statistical analysis. GN provided critical revision of the study results and intellectual content and prepared manuscript. All authors gave their final approval to the submitted manuscript.
Funding This work was supported by the National Science Centre (Grant Nos. NN404087940 and N404042/32/0945) and carried out with the use of the CePT infrastructure financed by the European Union-the European Regional Development Fund within the Operational Program (Innovative economy for 2007-2013).

Conflict of interest Authors declared no conflict of interest.
Ethical approval Subjects enrolled in the study were volunteers from the Outpatient Clinic of Metabolic Disorders of the National Food and Nutrition Institute. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Local Ethics Committee at the National Food and Nutrition Institute.
Informed consent Written informed consent was obtained from all of the registered volunteers. Declarations of consent in the study from all participants are available from correspondence author. Draft of the written consent form: I voluntarily and consciously agree to participate in the study. I read the information I received a copy of it. The goals and methods of the study were explained to me. I know that the study will carry out DNA analysis (carrier of genetic information) isolated from blood. I agree to perform DNA tests whose results will be used only for scientific papers. I know that participation in the study is voluntary and that I can resign from participation in the study at any time. I also declare that I have been informed that in accordance with the Act on the protection of personal data, the results of the study will not be used for other purposes than the abovementioned and that they will be kept in a place that prevents access to other people than those responsible for the above research.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.


extfieldsofstudy: Medicine Chemistry

text:
Development of hypoallergenic variants of the major horse allergen Equ c 1 for immunotherapy by rational structure based engineering

The use of recombinant allergens is a promising approach in allergen-specific immunotherapy (AIT). Considerable limitation, however, has been the ability of recombinant allergens to activate effector cells leading to allergic reactions. Recombinant hypoallergens with preserved protein folding and capacity to induce protective IgG antibodies binding effectively to the native allergen upon sensitization would be beneficial for safer AIT. In this study, hypoallergen variants of the major horse allergen Equ c 1 were designed by introducing one point mutation on the putative IgE epitope region and two mutations on the monomer-monomer interface of Equ c 1 dimer. The recombinant Equ c 1 wild type and the variants were produced and purified to homogeneity, characterized by size-exclusion ultra-high performance liquid chromatography and ultra-high resolution mass spectrometry. The IgE-binding profiles were analyzed by a competitive immunoassay and the biological activity by a histamine release assay using sera from horse allergic individuals. Two Equ c 1 variants, Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) showed lower allergen-specific IgE-binding capacity and decreased capability to release histamine from basophils in vitro when using sera from six allergic individuals. Triple 3 showed higher reduction than Triple 2 in IgE-binding (5.5 fold) and in histamine release (15.7 fold) compared to wild type Equ c 1. Mutations designed on the putative IgE epitope region and monomer-monomer interface of Equ c 1 resulted in decreased dimerization, a lower IgE-binding capacity and a reduced triggering of an allergic response in vitro.

produce antibodies which bind to natively folded biologically active proteins 3 . One approach is to use a peptide carrier strategy which has been reported to induce allergen-specific antibodies that recognize also the peptides in the context of the complete allergens 4 .
IgE epitopes are conformational consisting usually several non-continuous stretches (segments) of the polypeptide chain forming a specific three-dimensional structure on the surface of the allergen. The most accurate method to determine an IgE epitope is to co-crystallize the allergen in a complex with the Fab-fragment of patient-derived IgE-antibody 5 . Due to rather challenging techniques associated with isolation of allergen specific IgE antibodies and co-crystallization of protein complexes, only two such IgE/Fab-allergen complex structures have been determined so far; for bovine milk allergen Bos d 5 and for timothy grass pollen allergen Phl p 2. Interestingly, in both of the studies the overall spatial shape of the IgE epitope was flat consisting mainly of secondary structure elements (β-strands) differing from IgG epitopes which are typically located in the protruding regions of allergens 6,7 .
The immunocomplex structure of Bos d 5 revealed also that the allergen formed a dimer to which two identical Fab fragments were simultaneously bound indicating that two monoclonal IgE antibodies would be able to form a productive complex with a dimeric allergen to active mast cell or basophil 6 . In our subsequent studies involving a number of allergens with their crystal structures and mass spectrometric analyses, we have discovered that majority of the allergens are able to form weak or transient dimers depending on the allergen concentration 8 . We have proposed a sequential model for mast cell or basophil activation in which monomeric allergen binds first to the FcεRI bound IgE on the cell surface and subsequent dimerization is strongly boosted due to dimensional reduction (delocalization) on the cell surface leading to a signal transduction 9 .
The strategy to design hypoallergenic variants which have a diminished capability to form allergen-IgE complexes on the surface of an effector cell would thus include mutations on the flat IgE epitope and/or monomer-monomer interface. We have implemented these ideas in designing of two hypoallergenic variants of the major horse allergen Equ c 1. Horse allergy occurs in people who regularly work with horses, either professionally or for recreational purposes, and also in people indirectly exposed to horses. The prevalence of horse sensitization in occupational settings varies between 3.6 and 16.5% and people with a respiratory allergy have about

Results
Design of hypoallergenic variants. Mutations were designed both on the monomer-monomer interface and the putative IgE epitope of Equ c 1. The three-dimensional coordinates of Equ c 1 wild type were downloaded from the protein data bank (code 1EW3). The crystal structure contained one molecule in an asymmetric unit. The other half of the Equ c 1 dimer was formed by using the crystallographic symmetry operator. The extensive monomer-monomer interface (area 1024 Å 2 ) was studied by testing different mutations in the middle of the interface in silico. The effect of mutations were predicted by evaluating their ability to break molecular surface complementarity and hydrophobic interactions as well as hydrogen bonds and charged interactions between protein molecules. Hydrophobic groups do not interact well with polar groups, especially with charged ones. Therefore, substitution of hydrophobic residues with charged residues or vice versa should have more substantial effect to diminish protein-protein interaction. It was concluded that V110E and F112K mutations would be the most effective to disrupt the dimer formation of Equ c 1. The IgE epitopes of Equ c 1 are undefined, hence the putative IgE epitope was deduced by searching flat areas on the molecular surface of either side of the two-fold axis of symmetry of the Equ c 1 dimer by using PyMOL. Similarly, it was predicted that mutations E21Y and V47K would be the most promising candidates to achieve the decreased binding of IgE antibodies to Equ c 1. Two variants were selected for further studies: Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) ( Fig. 1A-D). Triple 1 variant with mutations G20V (putative IgE-epitope) combined to V110E and F112K was also designed, expressed in E.coli and purified with similar protocols as Triple 2 and 3. Expression level of Triple 1 was good, but it was much more prone to precipitate during adjustment of pH and concentration steps compared to Triple 2 and 3 and thus was not characterized further.
Mass spectrometry of rEqu c 1 wt and the hypoallergenic variants. The high-resolution mass spectrometric characterization of wild-type allergen and the variants Triple 2 and Triple 3 in denaturing solution conditions was used to determine the accurate molecular masses and to observe possible modifications in the protein preparations (Figs. 2A,C and S2A, SDS-PAGE analysis of the purifed Equ c 1 allergens is also shown in Fig. S3). The experimentally determined molecular mass (most abundant isotopic mass, averaged over the charge state distribution) of cytoplasmic rEqu c 1 wt was 20,166.04 ± 0.01 Da, which corresponds well with the theoretical molecular mass (20,165.99 Da) of the allergen with a disulfide bridge between Cys68 and Cys161. Similarly, the molecular masses of cytoplasmic Triple 2 (20,206.03 ± 0.04 Da) and Triple 3 (20,211.02 ± 0.04 Da) corresponded precisely with the theoretical masses calculated from the amino acid sequences of the hypoallergenic variants www.nature.com/scientificreports www.nature.com/scientificreports/ (20,206.02 Da and 20,211.01 Da for Triple 2 and Triple 3, respectively). The r Equ c 1 wt and Triple allergens were detected to be highly pure and homogeneous.
Native mass spectrometry was used to study the oligomeric state of proteins and to investigate the correct folding of the hypoallergenic variants compared with rEqu c 1 wt. The native MS showed that at the protein concentration of 40 μM, the rEqu c 1 wt allergen predominantly exists as a dimeric form, whereas the rEqu c 1 variants are mainly monomeric. The native ESI FT-ICR mass spectra displayed a limited number of low charge states, indicating that both the wild-type allergen and the variants have similar, tightly folded structures (Fig. 2B,D).
To quantify dimerization of rEqu c 1 wt, monomer-dimer ratios were measured over a range of allergen concentrations (0.1-4.8 μM) by using ESI MS. The fitted curve of the free monomeric allergen concentration against the total allergen concentration is shown in Fig. 1F. The calculated dissociation constant K d for rEqu c 1 wt dimer was 15.6 ± 0.8 nM which indicates strong tendency for Equ c 1 to dimerize but showing also a nature of this allergen to dissociate to monomers at nanomolar concentrations. It was not possible to perform similar measurements at different concentrations for Triple 2 and 3 because the signal for dimer was very low. However, by using intensities of monomer and dimer of Triple 3 at a single concentration (Fig. 2D), it can be estimated that K d for Triple 3 dimer would be roughly 0.8 mM.
IgE binding analysis. The serum IgE binding to the recombinant Equ c 1 wild type and Triple variants was analyzed by a miniaturized competitive immunoassay (Fig. 3). The wild-type Equ c 1 was used to capture anti-Equ c 1 IgE antibodies from a human serum sample as increasing amounts of soluble allergen (wt, Triple 2 or Triple 3) was used to inhibit the binding. Soluble allergen competed the binding of IgE antibodies to the immobilized wild type Equ c 1. The binding of serum IgE was analyzed from five horse allergic individuals and one non-allergic individual. The obtained data allowed us to calculate the half maximal inhibitory concentrations (IC 50 ) by fitting a curve to the dilution points of the dilution series of the competing allergen. The trend in the resulting curves were similar irrespective of the spotted concentration used. The results presented here are derived with the spotted wild type allergen concentration of 95 µg/ml.
The calculated IC 50 values for rEqu c 1 wild type were similar for all sera with the median value of 35 pM ( Table 1). The analyses showed reduced binding of allergen-specific IgEs for Triple variants 2 and 3 compared to rEqu c 1 wt (Fig. 3), medians 59 pM and 191 pM, respectively. The reduction was more significant for Triple 3 in all five serum samples. The results showed statistically significant differences between the two variants and wild type Equ c1 in three out of the eight comparisons (Table 1).
Histamine release of rEqu c 1 allergens. The biological activity of rEqu c 1 Triple variants 2 and 3 compared to the rEqu c 1 wild type was analyzed by HRA (Fig. 4). Histamine release was measured after passive

Discussion
ige epitope. We obtained very low IC 50 values (21 to 56 pM) for rEqu c 1 wild type in a competitive immunoassay analysis. These values reflect strong binding of Equ c 1 to specific IgE antibodies of serum and is in agreement with previous studies which showed that IgE antibodies exhibit stronger binding to allergens (K D 10 to 100 pM) compared to IgG binding to antigens (K D 10 to 100 nM) 6 . Both Triple 2 and Triple 3 variants showed consistently higher IC 50 values compared to wild type, median values being 59 and 191 pM, respectively, except for the serum E which showed similar IC 50 values between Triple 2 and wt. Reduced binding of allergen-specific IgEs to Triple variants 2 and 3 compared to wt allergen suggests that the mutations V47K (in Triple 2) and E21Y (in Triple 3) are located in the IgE epitope area of rEqu c 1 allergen. E21 seems to be more critical for IgE binding because its mutation produced consistently lower binding compared to V47 mutation. Both residues are located www.nature.com/scientificreports www.nature.com/scientificreports/ on the flat surface of the antiparallel β-sheet of Equ c 1 (Fig. 1B,D). The differences in the results of the competitive immunoassay may reflect heterogenicity of IgE-response among allergic individuals. This kind of flat or planar shape of an IgE epitope has previously been determined for Bos d 5 and Phl p 2 allergens from the immunocomplex structures between the Fab fragment of an IgE antibody and allergen 6,7 . Because mutations on this surface caused reduction in the IgE binding of sera from five different individuals, it could be argued that this deduced epitope located around V47 and E21 would be common or even generic for an IgE response among horse allergic individuals. Many recent studies have indicated that IgE response utilizes limited germline gene repertoire following somatic hypermutation and affinity maturation [15][16][17] . It is notable that different individuals seem to utilize similar type of sequences. This implicates that the number of IgE epitopes on the surface of an allergen is more limited compared to the number of IgG epitopes. Especially in the case of small allergens, as Equ c 1, dimer formation provides an optimal orientation of the IgE epitope(s) for recognition and binding by the IgE antibody-FcεRI complex on the effector cell surface 18 . It has been earlier shown by Lascombe et al. 14 that IgE binding of a serum pool of eight horse allergic individuals to Equ c 1 allergen was efficiently inhibited with only one anti-Equ c 1 monoclonal antibody mAb 220 indicating also that number of IgE epitopes of Equ c 1 is restricted.
Dimerization. One of the benefits of using native MS for studying oligomers is the detection of all oligomeric forms at the same time which is especially beneficial if protein oligomerization is transient and concentration dependent. Size-exclusion chromatography allows the separation of molecules in aqueous non-denaturing conditions based on their molecular size and shape, therefore enabling the separation of protein monomers from their dimers or aggregates without disrupting protein interactions. The native mass spectrometry and SE-UHPLC analyses indicate that rEqu c 1 wt exists predominantly in dimeric form in micromolar and higher concentrations but is able to dissociate to monomers at lower concentrations. These results are in agreement with the results of Gregoire et al. 19 who used ultracentrifugation and gel-filtration chromatography and found the strong tendency for Equ c 1 to form dimer but also reported the dimers can be dissociated to monomers by decreasing the protein concentration or lowering the pH of the buffer solution. The strategy to introduce two alterations on the hydrophobic monomer-monomer interface by replacing two hydrophobic amino acid residues by charged amino acid residues proved to be successful since the dimer/monomer ratio of Equ c 1 was dramatically decreased in Triple 2 and Triple 3 as shown by SE-UHPLC and native mass spectrometric analyses. The very narrow charge state distribution in the native mass spectra shows that Triple 2 and Triple 3 are still very compactly folded. The used monomer mutations would thus be effective in the reduction of cross-linking of specific IgE-allergen complexes on the surface of basophils or mast cells.
Histamine release assay. EC 50 values calculated from HRA results were extremely low, 5 to 45 pM (median 30 pM) for rEqu c 1 wt indicating that very tiny amount of this major horse allergen can trigger allergic reaction.
EC 50 values has been previously reported in some degranulation studies. Blanc et al. 20 have reported picomolar median values for peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6, namely 150, 2, 65 and 3 pM, respectively. On the other hand, major birch pollen allergen Bet v 1 seems to trigger degranulation at the higher concentrations, approximately in 2 nM 21 . This may indicate differences in the capacity of natural allergens to cross-link IgEs on the surface of basophils.
The low EC 50 values (5 to 45 pM, median 30 pM) obtained for rEqu c 1 wt is in agreement with low IC 50 values obtained from the competitive inhibition study (21 to 56 pM, median 35 pM), which explains that the potency of rEqu c 1 wt to trigger degranulation at extremely low concentrations is largely based on a strong binding of IgE antibodies to allergen. On the other hand, the increase in IC 50 values was smaller for Triple 2 (1.7 fold increase) and Triple 3 (5.5 fold increase) compared to EC 50 values for Triple 2 (7.7 fold increase) and Triple 3 (15.7 fold increase). Because IC 50 values reflects the binding of IgE antibodies, the higher increase in EC 50 values can be explained by two mutations (V110E + F112K) on the monomer-monomer interface of Equ c 1. Altogether, three point mutations, one on the putative IgE epitope and two on the monomer-monomer interface of Equ c 1 have thus successfully reduced the formation of dimeric allergen-IgE complexes and subsequent cross-linking of IgE on the cell membrane of basophils.

Recombinant hypoallergen candidates for AIT. As reviewed recently by Valenta et al. 1 , recombinant
allergen molecules have several advantages over natural extracts in AIT. In addition, it has been demonstrated that full length folded proteins are more effective in developing antibodies that recognize native structures 3 . It can be thus argued that natively folded recombinant allergen molecules or their derivatives with preserved native structure would be promising candidates in AIT. Phase II studies have been reached with recombinant wild type allergens from birch (Bet v 1) and timothy (Phl p 1, Phl p 2, Phl p 5, Phl p 6) 1 . The results have been positive but not so much superior that these products would have been further developed into market.
The key issues in the development of novel recombinant allergens or their derivatives for AIT are their efficacy and safety. One pathway forward to create a safe and efficient hypoallergen is to mutate a limited number of amino acids in allergen affecting its allergenicity yet preserving the genuine folded three-dimensional structure. Thus, the release of allergy mediators from mast cells and basophils and the consecutive appearance of allergic symptoms would be reduced. Instead, protective IgG antibodies against the wild type allergen would be induced. This would prevent mast cell and basophil activation and lead to the modulation of immunoresponse. However, this requires information on critical amino acid (hot spot) residues which especially contribute to the productive formation of allergen-IgE complex on the surface of mast cell or basophil.
Equ c 1 Triple 3 variant that contains three point mutations, one on the putative IgE epitope and two on the monomer-monomer interface, of the major horse allergen Equ c 1 has resulted in reduction of histamine release when using sera from five horse allergic patients. The mutations are not located in the region which was www.nature.com/scientificreports www.nature.com/scientificreports/ previously reported to include two major T-cell epitopes 12 . Amino acid sequence identity between Equ c 1 Triple 3 and wild type is extremely high, 98.4%, which considerably increases the possibility that IgG antibodies induced by Triple 3 would efficiently bind to Equ c 1 wt. Additionally as shown here, the Triple 3 was recombinantly produced and purified resulting in high purity, homogeneity and correct fold to support the steps toward consistent batch-to-batch manufacturing and safety of Triple 3 as medicinal product. Altogether this would make Triple 3 variant promising candidate for AIT in horse allergy. To evaluate the suitability of Triple 3 further for clinical studies, it would be important to test IgE-binding and histamine release with a wider panel of serum samples of individuals allergic to Equ c 1, to perform T-cell reactivity tests, and continue to animal studies. Recombinant Equ c 1 wt and Triple variants were produced in 1.8 L (6 × 300 ml) shake flask cultivations in LB medium supplemented with kanamycin (25 µg/ml) and cultivated at +37 °C with 220 rpm shaking until OD 600 reached ~1. Protein expression was induced by addition of IPTG (isopropyl β-D-1-thiogalactopyranoside) to a final concentration of 1 mM, and cultivation was continued for 4 h at +37 °C with 220 rpm shaking. Cells were harvested by centrifugation (15 min, 5000 rpm. +4 °C). The insoluble protein fraction was isolated from the cell pellet according to Hoffmann-Sommergruber et al. 23 and refolding of the allergen polypeptides according to Arango et al. 24 and Stancombe et al. 25 Briefly, collected bacterial cells were resuspended in 25 mM imidazole, 0.1% TritonX-100 pH 7.4 and lysed by three cycles of freezing and thawing steps. Inclusion bodies were isolated by centrifugation and solubilized in 6 M Urea, 10 mM CAPS pH 10.5 followed by a 3-hour incubation at room temperature under slow stirring. Urea was removed by slowly diluting the solubilization solution 1:10 (v/v) in 10 mM CAPS pH 10.5, after which pH was adjusted to 7.5. Protein was allowed to refold for minimum of 66 hours at +4 °C before concentration with Prep/Scale-TFF 6 ft2 10000 MWCO Cartridge (Millipore).

Design of variants.
Recombinant Equ c 1 wt and Triple variants were purified by a standard ion exchange (wild type with HiTrap DEAE followed by a HiTrap CM and variants with a HiTrap Q, GE Healthcare) followed by a size-exclusion chromatography (HiLoad Superdex 75 pg, GE Healthcare). Elution peak fractions after final purification step were analyzed by a Coomassie-stained SDS-PAGE showing that obtained proteins were of high purity and homogeneity. Concentrations of the pure allergens were determined by measuring A 280 and using sequence-derived extinction coefficient.

SE-UHPLC analysis.
Size-exclusion ultra-high-performance liquid chromatography (SE-UHPLC) analysis of rEqu c 1 wt and Triple variants were performed using Acquity BEH125 SEC column with dimensions of 4.6 × 150 mm, a pore size of 125 Å and a particle size of 1.7 µm (Waters) coupled with Acquity I-Class UPLC instrument (Waters) and controlled by Empower 3 software (Waters). The column was equilibrated to running conditions with PBS (12 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.3) as a mobile phase at a flow rate 0.3 ml/min until the baseline was stable. Samples of 2 µl of a 40 µM protein solution were injected. The chromatographic separation was carried out under isocratic flow with overall run time of 12 minutes and detection at 214 nm wavelength. The BEH125 SEC Protein Standard Mix (Waters) was used as a gel filtration standard.
Mass spectrometry. The mass spectrometric analyses were carried out with a 12 T SolariX XR FT-ICR mass spectrometer, equipped with an Apollo II ion electrospray ionization (ESI) source (Bruker Daltonics, Bremen, Germany). Protein samples were desalted and exchanged into an ESI-compatible ammonium acetate buffer (10 mM, pH 6.9) using PD-10 desalting columns (GE Healthcare). For mass spectrometric characterization in denaturing solution conditions, samples were diluted to the concentration of 0.2 μM with acetonitrile/water/ acetic acid solution (49.5:49.5:1.0, v/v). The protein samples were directly infused into the ion source by a syringe pump at a flow rate of 2 μl min −1 . The temperature and flow rate of the drying gas (N 2 ) were 200 °C and 4.0 L/min, respectively, and the pressure of the nebulizer gas was 1.0 bar. Mass spectra were acquired in the positive-ion mode over the m/z range of 800-3000 with 1-Mword time-domain transients and 0.05 s ion accumulation time.
For studying proteins in their native state, the instrumental parameters were carefully optimized to maintain the weak, non-covalent interactions in the gas-phase. Proteins were measured in 10 mM ammonium acetate buffer (pH 6.9). The drying gas temperature was set to 80 °C, and ion accumulation time was 1.0 s. For a native mass spectrum, 100 co-added 262-kword time domain transient were collected and processed to 524-kword data. The wild-type allergen and its Triple variants were measured by using the same instrumental parameters, in order to avoid any bias between the samples. Mass spectra were calibrated externally with respect to the ions of ES Tuning Mix (Agilent Technologies, Santa Clara, CA, USA). Data collection was controlled by ftmsControl software and the data were processed by using Data Analysis software ( www.nature.com/scientificreports www.nature.com/scientificreports/ dimers to monomers was taken in consideration by letting the diluted samples to stay four days at +4 °C. The actual concentration of the diluted samples was calculated just before the mass spectrometric analysis by the measured absorbance at 280 nm, using the sequence-derived extinction coefficient. The most diluted samples with concentrations of 0.1 and 0.25 μM showed very low absorbances, suggesting that the protein molecules were partially adsorbed to the walls of the plastic eppendorf tubes. For the less diluted protein samples, five parallel native mass spectra were measured at each different protein concentrations. The utilization of native ESI-MS for determining monomer-dimer ratio of (transient) protein complexes has been discussed in literature 26 . Briefly, the dissociation constant (K d ) was determined from the absolute intensities of ions representing either the monomer (P) or the dimer (P 2 ). The total intensities for P and P 2 were calculated by combining the peak intensities over the charge state distribution (z = 1 to n) in the ESI-MS spectra. The intensity ratio sufficiently reflects the concentration ratio in solution, if the ionization and transmissions efficiencies are similar for the monomer and dimer. K d for rEqu c 1 wt dimer was obtained using the procedure described in detail by Niemi et al. 9 .
Competitive IgE binding assay. For the competitive IgE binding assay rEqu c 1 wild type was biotinylated according to the manufacturer's instructions using a 10-fold molar excess of EZ-Link Sulfo-NHS-LC-Biotin reagent (ThermoFisher Scientific). The biotinylated Equ c 1 wt was spotted as three concentrations (190, 95 and 47.5 µg/ml, 1 nl/spot) onto the streptavidin coated microtiter plate wells (ThermoFisher Scientific) by Nano-Plotter 2.1 (GeSiM). Serum samples from five horse allergic individuals were selected based on their ImmunoCAP ISAC (ThermoFisher Scientific) test profiles. The serum samples used in the competitive immunoassay and histamine release assay were collected from volunteer horse allergic adult (over 18 years) individuals after the permission of the Ethical Committee of Helsinki University Hospital (Finland). The optimal dilution for each serum chosen to be used in the competitive assay was analyzed by IgE serum titration analysis for each spotted concentration (data not shown). For competitive assay, diluted serum was pre-incubated with different amounts of competing allergen in final allergen concentrations of 10 000, 2 000, 400, 80, 16, 3, 0.6 and 0 pM and then added to the well in which the biotinylated rEqu c 1 wt was immobilized. All of the experiments were performed in duplicates.
Peroxidase-conjugated anti-human IgE (Southern Biotech) was used for secondary detection and fluorescently-labelled tyramide (ThermoFisher Scientific) for signal amplification. The fluorescence readout was obtained by scanning the wells with LS400 microarray scanner and 633 nm laser (Tecan). Image analysis, spot detection and fluorescence intensity quantification were performed with Array-Pro Analyzer software (Media Cybernetics). Local background signal value was measured outside the specific spots from each well. The net signal value for each spot was obtained by subtracting the local background signal value from the average intensity value of each spot.
Histamine release assay. Biological activity of the rEqu c 1 wild type and Triple variants was analyzed by a histamine release assay (HRA). HRA was performed as an outsourced service at RefLab Aps (Copenhagen, Denmark). Briefly, stripped human basophils were passively sensitized with rEqu c 1 wt and Triple variants in 16 different concentrations from 0.05 fM to 0.5 nM (1fg/ml to 10 ng/ml) using the same five serum samples (A-E) as in the competitive IgE binding assay and with an additional positive control serum sample (F) provided by RefLab. Released histamine induced by different allergen concentrations was measured as duplicates by ELISA (enzyme-linked immunosorbent assay) using a glass microfibre method developed by RefLab. In this method glass microfibres coated on microtiter wells bind histamine released from allergen triggered basophils with high affinity and selectivity 27 . Statistical analysis. Graphical display, curve fitting, Student's t-test analysis of competitive ELISA and histamine release assay data were done with GraphPad Prism v. 8.2.1 (GraphPad Software Inc.). A P value of less than 0.05 was considered statistically significant. Statement of using human sera. Human participants are not directly involved in this study. The origin of the human serum samples used in the competitive immunoassay and histamine release assay (HRA) and the human basophils utilized in HRA is described as follows. The serum samples used in the competitive immunoassay and histamine release assay were collected from volunteer horse allergic individuals after the permission of the Ethical Committee of Helsinki University Hospital (Finland). Serum samples were collected by Desentum Ltd as an outsourced service using healthcare professionals. The blood samples were numbered and the identity of the donors is known only by the responsible scientist and the healthcare professionals who participated in the collection of the blood samples. Biological activity of the recombinant allergens was analysed by the histamine release assay as an outsourced service at RefLab ApS (Copenhagen, Denmark). RefLab is an accredited laboratory according to: EN ISO/IEC 17025: 2005 and has an in house assay for measuring histamine release from basophils obtained from blood bank buffy coats. Buffy coats were collected at the Blood Bank, National University Hospital of Copenhagen. The Blood Bank has a general ethical approval to hand out buffy coats making sure that the blood donors are anonymous. It is therefore not possible to obtain case history or laboratory case records of the blood donors. RefLab has a contract with the Blood Bank allowing using the buffy coats. The study was conducted in compliance with the Declaration of Helsinki.