This is the peer reviewed version of the following article: New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7 Carmen Oeo-Santos, Ana Navas, Sara Benedé, Berta Ruíz-León, Araceli Díaz-Perales , Lothar Vogel, Carmen Moreno-Aguilar, Aurora Jurado, Mayte Villalba, Rodrigo Barderas. Allergy. 2020 Apr;75(4):798-807. which has been published in final form at https://doi.org/10.1111/all.14086 For Peer Review 90x60mm (300 x 300 DPI) Page 6 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review New insights into the sensitization to non-related nsLTPs from pollen and food: new role of the allergen Ole e 7 C. Oeo-Santos, A. Navas, S. Benedé, B. Ruíz-León, A. Díaz-Perales, I. Vogel, C. Moreno- Aguilar, A. Jurado, M. Villalba and R. Barderas. 1. Ole e 7 cross-reacts with LTPs from pollen and food, specifically with peach and pear. 2. Common IgG and IgE epitopes were identified between Ole e 7 and Pru p 3 despite their low amino-acid sequence identity. 3. Ole e 7 could act as primary sensitizer in regions with high olive-pollen exposure, leading to Pru p 3 sensitization. Page 7 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 1 1 New insights into the sensitization to non-related nsLTPs from pollen and food: 2 new role of the allergen Ole e 7. 3 4 Carmen Oeo-Santos, BSa*, Ana Navas, BS b, c*, Sara Benedé, PhDa, Berta Ruíz-León, MD, 5 PhD b, c,d, Araceli Díaz-Perales, PhDe,d, Lothar Vogel, PhDe, Carmen Moreno-Aguilar, MD, 6 PhD b, c,d, Aurora Jurado, MD, PhD b, c,d, Mayte Villalba, PhD a,d,^, Rodrigo Barderas, 7 PhDf,^. 8 9 * Both authors contributed equally. 10 11 a Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 12 Universidad Complutense de Madrid, 28040 Madrid, Spain. 13 b UGC Inmunología y Alergia, Hospital Universitario Reina Sofía de Córdoba, 14004 Córdoba, 14 Spain. 15 c Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/ Hospital 16 Universitario Reina Sofía/ Universidad de Córdoba, 14004 Córdoba, Spain. 17 d Allergy Network ARADyAL. Instituto de Salud Carlos III, Madrid, Spain. 18 e Centro de Biotecnología y Genómica de Plantas (CBGP, UPM-INIA), Campus de 19 Montegancedo-UPM, 28223 Madrid, Spain. 20 f Division of Allergology, Paul-Erlich-Institut, 63225, Langen, Germany. 21 g UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, Majadahonda 28220, 22 Madrid, Spain. 23 24 ^ To whom correspondence should be addressed: 25 Rodrigo Barderas. 26 Functional Proteomics Unit, UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, 27 E-28222 Majadahonda, Madrid, Spain; Tel.: 34-91-8223231; E-mail: r.barderasm@isciii.es 28 29 ^ To whom correspondence should be addressed: 30 Mayte Villalba. 31 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 32 Universidad Complutense de Madrid, E-28040 Madrid, Spain; Tel.: 34-91-3944155; E-mail: 33 mvillalb@ucm.es 34 35 Abstract Word count: 247 36 Text Word count: 3865 Page 8 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2 37 Abstract 38 Background: Ole e 7 is a non-specific lipid transfer protein (nsLTP) from olive pollen, one 39 of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe 40 symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized 41 patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no 42 evidence of cross-reactivity which explains this observed co-sensitization. Therefore, the 43 purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. 44 Methods: A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the 45 study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. 46 Inhibition assays were performed to determine the existence of cross-reactivity between 47 both nsLTPs. Allergic response was analyzed ex vivo (Basophil Activation Test) and in 48 vitro (RBL-2H3 mast cell model). 49 Results: Common IgG and IgE epitopes were identified between both allergens. IgE- 50 binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as 51 inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a 52 response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. 53 Conclusions: Our results suggest that Ole e 7 could play a new role as primary sensitizer 54 in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This 55 co-sensitization process would occur because of the cross-reactivity between Ole e 7 and 56 Pru p 3 observed in some allergic patients. 57 58 Short title 59 New insights into non-related nsLTPs sensitization 60 61 Keywords 62 Cross-reactivity, Non-specific Lipid Transfer Protein, Olive pollinosis, Peach allergy, 63 Primary sensitization. 64 65 Abbreviations 66 BAT- Basophil Activation Test 67 ELISA- Enzyme-Linked ImmunoSorbent Assay 68 nsLTP- Non-specific Lipid Transfer Protein 69 SPT- Skin Prick Test 70 SEM- Standard Error of the Mean 71 WB- Western Blotting Page 9 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 3 72 Introduction 73 The huge development of proteomics and molecular biology techniques in the last years 74 has boosted the identification of specific proteins responsible for the allergic responses with the 75 aim to improve the diagnosis and immunotherapy protocols of the pathology. The disease 76 exacerbation is dependent on the severity of allergen-specific symptoms or whether allergy is 77 caused by a genuine sensitization (primary or species-specific) or by cross-reactivity (1). Cross- 78 reactivity is due to the existence of IgEs, which recognize the same epitope in allergens from 79 different biological sources (2). These reactions have been widely described among many 80 allergenic protein families, including pollen and food allergens (3-5). 81 Non-specific Lipid Transfer Proteins (nsLTPs) comprise a protein family of 7-9 kDa, 82 widely distributed throughout the plant kingdom (6). Notably, several members of the nsLTP 83 family are considered main allergens in the Mediterranean area. Among them, Pru p 3, the 84 major allergen from peach, is one of the most frequently recognized fruit allergens and plays a 85 relevant role in cross-reactivity, even with pollen LTPs (7-10). Additionally, Pru p 3 behaves as 86 the primary sensitizer in the development of food allergy to Rosaceae species in regions where 87 the presence of birch is rare and the involvement of Bet v 1-like allergens is infrequent, such as 88 in southern Europe (3, 11-13). However, it has been described that Pru p 3 could lose its 89 primary sensitizer role in regions where birch is scarce and the presence of other pollens is 90 intense (14, 15). 91 Olive tree pollinosis is worldwide increasing due to its extensive cultivar. Indeed, it is a 92 main allergenic source in California (US), China, India, Australia and South America beyond 93 the Mediterranean Basin, where this pollen has been extensively studied (16). Fourteen olive 94 pollen allergens have been identified so far (16-18). Among them, the nsLTP Ole e 7 is a major 95 allergen associated to severe clinical symptoms in regions with high levels of olive pollen 96 counts such as in Andalusia (Spain) (19, 20). In this area, some allergic patients to Ole e 7 show 97 clinical symptoms to Rosaceae fruits, being the co-sensitization to Pru p 3 frequently observed. 98 Despite the 31% of amino acid sequence identity reported between Ole e 7 and Pru p 3 99 (21), a co-sensitization to these two LTPs has been previously described in other Spanish Page 10 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 4 100 regions (22). Therefore, an immunological relationship between Ole e 7 and Pru p 3 may not be 101 excluded, with Ole e 7 as the primary sensitizer in the development of peach allergy, especially 102 in areas with high exposure to olive pollen. However, the underlying causes of this co- 103 sensitization have not yet been elucidated. 104 In this context, the aim of the present work was to explore the role of Ole e 7 in the 105 sensitization process to peach by means of Pru p 3, in those regions with olive pollen counts 106 over 5000 grains/m3, to get further insights into the underlying causes of a possible co- 107 sensitization. To this end, we performed in vitro analysis of the IgE response to both allergens 108 by ELISA and WB, followed by ex vivo and in vitro cellular response analyses in basophils and 109 the RBL-2H3 mast cell line model. 110 We reported for the first-time evidences of cross-reactivity between Ole e 7 and Pru p 3, 111 which had not yet been examined. Moreover, we identified four Ole e 7-monosensitized patients 112 who developed a cellular response against Pru p 3, despite their unique sensitization to Ole e 7 113 by ImmunoCAP 250 and SPT. Our results suggest that Ole e 7 could behave as a primary 114 sensitizer in regions with high olive pollen exposure, leading to a secondary sensitization to Pru 115 p 3 with or without symptoms. Page 11 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 5 116 Materials and Methods 117 See the materials and methods section in this article's supporting information online for full 118 details about protein extracts, allergens and antibodies, IgG-binding analysis, specific IgE 119 binding to Ole e 7 and Pru p 3, cell-based allergic mediator release assays, and ELISA and WB 120 inhibition assays. 121 122 Patients 123 The study was approved by the Ethical Committees of the Reina Sofía University 124 Hospital, Córdoba, Spain (ref. 3033), and the Complutense University and Instituto de Salud 125 Carlos III (CEI P49). Written informed consent was obtained from all patients. All samples 126 were anonymously handled. 127 A total of 48 patients recruited at the Immunology and Allergy Department of the Reina 128 Sofía University Hospital (Córdoba, Spain) with a confirmed history of allergy to olive pollen 129 with sensitization to Ole e 7 and/or sensitization to LTP from peach (Pru p 3) were included in 130 the study. Clinical evaluation included examination of patient history, SPT and determination of 131 specific IgE (sIgE). The SPT was performed according to the European guidelines (23), using 132 commercial extracts from Olea europaea pollen and peach (ALK-Abelló, Madrid, Spain). A 133 positive SPT response was considered when the diameter of the wheal was 3 mm greater than 134 that induced by the negative control. sIgE to Ole e 7 and Pru p 3 were measured by 135 ImmunoCAP 250 (Phadia, Uppsala, Sweden) according to the manufacturer recommendations. 136 Patients were stratified according to their sensitization status by ImmunoCAP 250 137 (Supporting Table 1). In addition, patients were also analyzed by SPT to O. europaea and Pru p 138 3. Accordingly, three groups of study were defined: 139 I. Ole e 7-monosensitized patients (n=13) had sIgE to Ole e 7 >0.35 kU/L and sIgE to Pru 140 p 3 <0.35 kU/L. In addition, a positive SPT response to O. europaea pollen and a 141 negative SPT response to LTP-peach were observed in all patients. 142 II. Pru p 3-monosensitized patients (n=7) had sIgE to Pru p 3 >0.35 kU/L and sIgE to Ole e 143 7 <0.35 kU/L. In addition, a positive SPT response to Pru p 3 was observed in 71.43% 144 of patients and a negative SPT to O. europaea pollen in all patients. Page 12 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 6 145 III. Ole e 7 and Pru p 3-bisensitized patients (n=28) had a sIgE level >0.35 kU/L to both 146 allergens. In addition, 60.71% of these patients showed a positive SPT response to Pru p 147 3 and 92.86% to O. europaea pollen. 148 Patients sensitized to Ole e 7 exhibited rhinoconjunctivitis and bronchial asthma due to the 149 exposure to olive pollen. Patients sensitized to Pru p 3 had a clinical history of allergic reactions 150 following the ingestion of Rosaceae fruits. Symptoms of food allergy included oral allergy 151 syndrome (OAS), urticaria/angioedema, gastrointestinal symptoms or anaphylaxis. None of the 152 patients had been treated with immunotherapy for the last three years before the inclusion in the 153 study or was being treated with active immunotherapy. Page 13 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 7 154 Results 155 Ole e 7 displays IgE-cross-reactivity with nsLTPs from food-derived extracts 156 Previous studies have described cross-reactions between several LTPs from different 157 biological sources. Nevertheless, the role of Ole e 7 in these processes has not been clarified due 158 to the low amount of natural Ole e 7 allergen obtained after its purification. The successful 159 expression of Ole e 7, whose primary amino acid sequence was obtained by proteomics and 160 retains the structural, allergenic and antigenic properties of the natural allergen (17), has enabled 161 a deeper analysis of the cross-reactivity of Ole e 7 with other LTPs. First, we surveyed different 162 pollen and food-derived extracts by an ELISA inhibition test to determine whether any LTP 163 could cross-react with Ole e 7. To this end, six pollen and ten food-derived extracts were used 164 as inhibitors of the IgE-binding to Ole e 7 (Fig 1A, B). Regarding pollen extracts, O. europaea 165 used as control of the assay was almost able to completely inhibit the IgE-binding to Ole e 7. In 166 addition, other members from the Oleaceae family showed significant inhibition values: 80% L. 167 vulgare from Ligustrum genus, 56% S. vulgaris from Syringa genus and 65% F. excelsior from 168 Fraxinus genus (Fig 1A). Moreover, the highest significant inhibition with food-derived extracts 169 was observed with peach (P. persica) and pear (P. communis) extracts as inhibitors, with 170 significant inhibition values of 67 and 52% respectively (Fig 1B). 171 These results suggest the existence of cross-reactions between Ole e 7 and LTPs from 172 pollen and food-derived extracts. 173 174 Ole e 7 and Pru p 3 share common IgG epitopes 175 We next focused the study on the potential LTP pollen-food cross-reactivity between 176 Ole e 7 and Pru p 3 because of their clinical relevance, even though they possess low amino acid 177 sequence identity (Supporting Fig 1). 178 Prior to establish structural relationships analyzing the IgG recognition of both LTPs, 179 the quality of the recombinant allergens was assessed by Coomassie blue staining and mass 180 spectrometry (Fig 2A, Supporting Fig 2). Then, the IgG-binding to each allergen from each Page 14 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 8 181 specific polyclonal antiserum to rOle e 7 or rPru p 3 was analyzed by WB and ELISA (Fig 2B- 182 D). 183 Although Ole e 7 was also barely recognized by the Pru p 3 polyclonal antiserum by 184 WB, Ole e 7 and Pru p 3 were recognized by both polyclonal antisera (Fig 2B). Then, the IgG- 185 binding to the native proteins was analyzed by ELISA (Fig 2C, D). In agreement with the 186 previous results obtained by WB, Pru p 3 polyclonal antiserum was able to recognize Ole e 7. In 187 contrast, although the specific Ole e 7 polyclonal antiserum slightly recognized Pru p 3, these 188 results show the presence of common IgG epitopes in both LTPs, which could also cause their 189 cross-reactivity at IgE level. 190 To address this question, our next aim was to study the existence of common IgE 191 epitopes, and further analyzed their potential IgE cross-reactivity by cellular assays. 192 193 Some patients may recogniz equivalent IgE epitopes from olive pollen and peach 194 nsLTPs. 195 Inhibition assays by ELISA and WB were performed using individual sera from two 196 Ole e 7-monosensitized and four bisensitized patients, whose sIgE levels were measured by 197 ELISA and ImmunoCAP (Supporting Fig 3, Supporting Fig 4 and Supporting Table 1). Both 198 proteins were used on the solid phase and as inhibitors (Fig 3, Supporting Fig 4). 199 Interestingly, the bisensitized patient 44 used as control of IgE reactivity to both LTPs 200 showed IgE-binding inhibition by ELISA as well as WB. By ELISA, a complete inhibition of 201 the IgE-binding to rOle e 7 was obtained using both rOle e 7 and rPru p 3 as inhibitors. In 202 contrast, inhibition to rPru p 3 was barely observed using rOle e 7 as inhibitor (Fig 3A, B). By 203 WB, a complete inhibition to the IgE-binding to rOle e 7 was achieved using rOle e 7 and rPru p 204 3 as inhibitors, and a 25% IgE-binding inhibition to rPru p 3 was reached using rOle e 7 as 205 inhibitor (Fig 3C, D). In addition, three other bisensitized patients were analyzed by ELISA 206 (Supporting Fig 4). For patients 1 and 27, Ole e 7 or Pru p 3 as inhibitors were almost able to 207 abrogate the IgE binding to each protein. On the other hand, similar results with that of patient 208 44 were also obtained for patient 21. These results indicate, apart from the heterogeneous IgE Page 15 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 9 209 reactivity of patients, that some IgE epitopes of Pru p 3 seem to be absent in Ole e 7, especially 210 in the native conditions of the protein. However, we cannot discard that these epitopes could not 211 be highly recognized by sIgEs from patients, being the affinity for those epitopes decisive to 212 identify the epitopes involved in cross-reactions. Even so, although further experiments would 213 be performed to determine the relevance of the different affinity of the IgE, the inhibition of the 214 IgE-binding observed in the experiments suggest the existence of common conformational and 215 linear epitopes supporting the cross-reactivity between both allergens. 216 Surprisingly, the two Ole e 7-monosensitized patients analyzed by WB showed an 217 inhibition of the IgE binding to rOle e 7 of 25 and 100%, using rPru p 3 as inhibitor (Fig 3E). 218 No IgE binding was detected to rPru p 3 strips by any patient. Furthermore, we explored the 219 differences on the IgE recognition to those epitopes under denaturing and non-denaturing 220 conditions. A reduction of the signal of about 15% was detected under non-denaturing 221 conditions (Fig 3F). The lack of conformational epitopes of the protein in the presence of β-ME 222 (denaturing conditions) suggests, besides the existence of common conformational epitopes 223 observed by ELISA with bisensitized patients, the existence of common linear IgE epitopes for 224 both allergens. 225 226 Analysis of the release of cell mediators reveals that Ole e 7 could trigger the allergic 227 response to the nsLTP from peach in Ole e 7-monosensitized patients. 228 Next, we proceeded to study this reactivity at IgE level in the forty-eight patients 229 sensitized to Ole e 7 and/or Pru p 3 according to ImmunoCAP 250 (Supporting Figure 3 and 230 Supporting Table 1) by means of ex vivo cellular assays by BAT (Figure 4, Supporting Figure 5) 231 and by in vitro analysis using the RBL-2H3 mastocyte cell model (Figure 5). 232 Regarding the Ole e 7 response by BAT (Fig 4A), we observed that the percentage of 233 degranulated basophils was significantly higher in Ole e 7-monosensitized and bisensitized 234 patients than in Pru p 3-monosensitized patients (p=0.001 and p<0.001, respectively), whereas 235 no significant differences were found between Ole e 7-monosensitized and bisensitized patients 236 (p=0.864). Interestingly, when considering the Pru p 3 response (Fig 4B), no significant Page 16 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 10 237 differences between the three analyzed groups were found. Surprisingly, we identified four Ole 238 e 7-monosensitized patients whose basophils degranulated after the incubation with rPru p 3. 239 Thus, while basophils from Pru p 3-monosensitized patients were not able to degranulate in the 240 presence of Ole e 7, basophils from 4 out of 13 Ole e 7-monosensitized patients were also able 241 to degranulate in the presence of Pru p 3. 242 To confirm the specificity of the observed response, BAT was performed at 0.1 µg/mL, 243 1 µg/mL and 10 µg/mL of rOle e 7 and rPru p 3 in the four Ole e 7-monosensitized patients who 244 responded to Pru p 3, and two random selected bisensitized patients. Clinical characteristics of 245 these patients are summarized in Table 1. Basophils from the four Ole e 7-monosensitized 246 patients (patient 9, 12, 16 and 36) degranulated similarly at the three concentrations of rOle e 7. 247 However, when rPru p 3 was used to promote degranulation, the response was only achieved at 248 the highest concentration. These results show that apart from rOle e 7, rPru p 3 can induce 249 basophil degranulation from Ole e 7-monosensitized patients. Basophils from the bisensitized 250 patient 37 behaved like the four-rOle e 7-monosensitized patients (Fig 4C, D), in line with the 251 serum sIgE levels obtained by ELISA, in which IgE was detected only with rOle e 7 252 (Supporting Fig 3). Regarding bisensitized patient 38, its degranulation percentage was similar 253 at the three different concentrations either with rOle e 7 or rPru p 3 as stimulus (Fig 4C, D). 254 Lastly, to further confirm these results, these patients sera were analyzed using the 255 RBL-2H3 mastocyte cell model, which allows the measurement of the release of ẞ- 256 hexosaminidase. Four different concentrations of allergen were used (Fig 5). Interestingly, 257 despite their unique sensitization to Ole e 7, all Ole e 7-monosensitized patients tested were able 258 to react with rPru p 3. Regarding bisensitized patients 37 and 38, although a similar reaction to 259 rOle e 7 was observed at the highest concentration of allergen, the response of patient 38 was 260 mainly produced with rPru p 3 as stimulus. In this patient, these results may suggest the role of 261 Pru p 3 as primary sensitizer. 262 Collectively, the herein presented results show the existence of common conformational 263 and linear IgE-binding epitopes in some allergic olive pollen patients that could explain the 264 pollen-food cross-reactivity observed in clinics between Ole e 7 and Pru p 3. Page 17 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 11 265 Discussion 266 Cross-reactivity between pollen and food LTPs have been extensively demonstrated, 267 although the identification of the primary inducer in these patients is still unclear (3, 7, 24, 25). 268 A common feature of LTP-allergic patients is their multiple plant-food sensitizations, which is 269 known as LTP syndrome, a high relevant hypersensitivity disorder in the Mediterranean basin 270 due to its association with severe symptoms, such as anaphylaxis (26, 27). nsLTPs from 271 artemisia (Art v 3) and plane tree (Pla a 3) pollens are highly implicated in this syndrome since 272 they cross-react with LTPs from different foods (28). Although this association has not been 273 described for Ole e 7 or Par j 1, which show low amino acid sequence similarity with nsLTPs 274 from foods (26), their role in the LTP syndrome is still unclear. On the other hand, the 275 sensitization to LTPs seems to be usually related to peach-specific IgE levels (26). In southern 276 Europe, the main allergen from peach -Pru p 3- is considered a primary sensitizer for the 277 development of allergy to other pollen and plant-food LTPs (10, 29). However, in regions with 278 high pollen exposure (i.e. mugwort or ragweed), Pru p 3 may lose its role as primary sensitizer 279 (14, 15). 280 We used in the study sera from patients from the south of Spain where olive tree is 281 widely cultivated and its pollen is the main cause of allergy in these populations. Co- 282 sensitization to Ole e 7 and Pru p 3 has been reported in several patients from this area (22, 30), 283 but no cross-reactivities between these two nsLTP have been described to date. Indeed, 284 preliminary results of our group using the natural Ole e 7 allergen isolated from pollen pointed 285 out to an absence of cross-reactivity (16, 31). In this sense, it is necessary to take into account 286 that the natural allergen isolated from pollen is a heterogeneous mixture of multiple isoforms 287 obtained in a very low concentration and that the experiments were carried out using pools of 288 several sera from allergic patients to Ole e 7. In this context, the main objective of the present 289 study was to clarify whether Ole e 7 is involved in cross-reactivity and which is its implication 290 in the sensitization process to other nsLTPs. Page 18 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 12 291 Here, we have demonstrated that Ole e 7 cross-reacts, not only with LTPs from pollens, 292 but also with LTPs from pear and peach (Pyr c 3 and Pru p 3, respectively) in spite of the low 293 sequence identity reported for them (Supporting Figure 1). The obtained results are in 294 agreement with a previous study which established a clinical association between severe food 295 allergic clinical symptoms and sensitization to Ole e 7 (32). Nevertheless, other studies do not 296 support that correlation (21, 33). The observed differences may be attributed to the 297 multifactorial and heterogeneous character of this disorder and the molecules involved in it. 298 Hence, the differences between the studied populations may be explained by the demographic 299 characteristics of each area linked to different environmental factors (34-36). Furthermore, we 300 have here confirmed the antigenic and allergenic cross-relations between Ole e 7 and Pru p 3. 301 Common IgG epitopes were observed although the signal detected for Ole e 7 by the specific 302 polyclonal antiserum against Pru p 3 was much lower than that for Pru p 3, which can be 303 directly related to the affinity of the IgG-binding or the number of recognized epitopes. 304 Common IgE epitopes were also identified. In addition, the conformation of the protein seems 305 to be crucial for the IgE-binding, being recognized mainly under reducing conditions as 306 confirmed by IgE inhibition WB assays. However, the elucidation of which IgE epitopes were 307 recognized in both allergens was not feasible because the 3D structure and the epitope map of 308 Ole e 7 are still unknown, in contrast to the deep analysis so far performed for Pru p 3, whose 309 IgE-binding epitopes have been well characterized (37). Further structural and immunological 310 studies of allergens from the LTP protein family are required to determine which are the main 311 epitopes recognized by allergic patients sensitized to different LTPs. 312 Remarkably, although an absence of recognition was observed by ELISA and WB, we 313 have identified Ole e 7-monosensitized patients whose basophils reacted against rPru p 3, which 314 was further supported using the RBL-H3 mast cell model. This could be due to the fact that 315 cellular assays are more sensitive than ELISA and WB; or alternatively because low affinity 316 epitopes not recognized by specific IgEs by ELISA or WB were able to trigger a cellular 317 response (38, 39). On the other hand, the absence of lipids in rPru p 3 could also partially affect 318 the detection of some sIgE levels from patients. In addition, none of the patients had clinical Page 19 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 13 319 symptoms to peach or other Rosaceae members, as well as some allergens whose co- 320 sensitization had been diagnosed (37). The obtained results make us to raise the possibility that 321 these patients are in an early stage of the sensitization process to Rosaceae (Pru p 3), acting Ole 322 e 7 as the primary LTP sensitizer promoted by the high olive pollen exposure in Andalusia, as it 323 was described for Artemisia pollen in other regions (3, 7, 14, 22). A similar hypothesis was 324 proposed to explain the linking between food allergen and grass (40), or birch pollinosis (41). 325 On the other hand, we cannot discard a different sensitization route by airways instead of the 326 gastrointestinal via described for food allergens sensitization. Indeed, Pérez-Calderón et al. 327 suggested that occupational allergy to peach arises as a consequence of the inhalation of Pru p 3 328 present in the leaves of the plant, and not as a result of fruit ingestion (42). In addition, the study 329 performed by Garcia et al. also supports these data. The authors also confirmed that Pru p 3 330 from peach leaves act as a respiratory allergen, causing occupational rhinoconjunctivitis and 331 asthma (43). Marzban et al. also raised this alternative mechanism for allergy development in 332 their study of the sensitization to birch pollen and Mal d 1 and Mal d 3 allergens (44, 45). The 333 influence of other cofactors should also be considered because they are frequently involved in 334 the development of the allergic response and in the clinical expression of allergic symptoms 335 (46). Among these factors, the mucosa and the microbiome could also play an important role. 336 The mucosa immune system regulates the entrance of multiple antigens either pathogenic or 337 non-pathogenic, such as food and pollen allergens. Interestingly, it has been described that in 338 spite of the absence of food allergy, the oral mucosa could be compromised due to a respiratory 339 sensitization (47). A disrupted barrier could be responsible for food allergy development, which 340 support our hypothesis about the sensitization process in some analyzed patients. On the other 341 hand, in food allergy, there have been reported differences in the oral and intestinal microbiome 342 that may increase the susceptibility to develop sensitization to food allergens (48). Therefore, 343 the microbiome of the oral mucosa, a shared entrance place between pollen and food allergens, 344 could influence the co-existence of different allergenic processes. Further studies to clarify the 345 mechanism of the co-sensitization between these two allergens are required for a better 346 diagnosis and treatment. Page 20 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 14 347 In conclusion, our study demonstrates the cross-reactivity between the nsLTP from 348 olive pollen and peach, a subject of debate for years. Furthermore, data herein presented provide 349 new insights regarding the sensitization process to fruit nsLTP, particularly to Pru p 3. Our 350 results suggest that Ole e 7 could act as a primary sensitizer in regions with high olive pollen 351 exposure, probably leading the peach sensitization by a new route, the respiratory tract. 352 However, further studies are needed to determine which factors are crucial in the onset of the 353 allergic symptoms to peach. 354 355 Author Contributions: Conception and design: C.O-S., A. N., C.M-A., A.J., M.V., R.B. 356 Development of methodology: C.O-S., A.N., S.B., L.V. Perform Research: C.O-S., A. N., S.B., 357 B.R-L., A.J. Analysis and interpretation of data: C.O-S., A. N., S.B., B.R-L., A.J., R.B., C.M., 358 M.V. Writing, review, and/or revision of the manuscript: C.O-S., A. N., A.J., C.M., M.V., R.B. 359 Technical, obtaining and processing of samples, or material support: A.J., L.V., C. M-A., A. D- 360 P., A.J., M.V., R.B. 361 362 Acknowledgements 363 This work was supported by SAF2014-53209-R and SAF2017-86483-R grants from the 364 Ministerio de Economía y Competitividad to R.B. and M.V., and to M.V., respectively, and 365 RIRAAF Network RD12/0013/0015 grant and ARADyAL Network RD16/0006 grant from the 366 Instituto de Salud Carlos III (ISCIII) co-founded by Fondo Europeo de Desarrollo Regional - 367 FEDER- for the Thematic Networks and Co-operative Research Centres. B.R., A.J., A.N, and 368 C.M. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the 369 Alergosur Foundation. We also gratefully acknowledge the financial support from the ISCIII for 370 the PI17CIII/00045 grant to R.B. S.B. was a recipient of the Juan de la Cierva programme of 371 the MINECO. We also thank the excellent technical support of Sara Abián. 372 373 Supporting Information 374 Supporting information to this article can be found online. Page 21 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 15 375 376 Conflicts of Interest Statement 377 The authors declare no conflicts of interest. Page 22 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 16 378 Tables O liv e po lle n se ns iti za tio n Pe ac h se ns iti za tio n P at ie nt S ex A ge (Y ) S ym pt om s (R C /A /R C A ) S P T Ig E O le e 7 (C A P ) S ym pt om s (N o/ O A S /U /A n) S P T Ig E P ru p 3 (C A P ) 9 F 41 R C A + 20 1 N o - 0 12 F 23 R C A + 42 .9 N o - 0 16 F 22 R C A + 58 .9 N o - 0 36 M 48 R C A + 12 5 N o - 0 37 F 18 A + 17 8 N o - 1. 13 38 M 22 A + 25 .8 O A S /U + 25 .8 44 F 44 R C A + 8. 71 O A S /U + 20 .8 T ab le 1 . C lin ic al c ha ra ct er is tic o f p at ie nt s s el ec te d to p er fo rm B A T a na ly si s. M , m al e; F , f em al e; R C , r hi ni tis ; A , a st hm a; R C A , r hi ni tis an d as th m a; N o, N o sy m pt om s; O A S, o ra l a lle rg y sy nd ro m e; U , u rti ca ria ; A n, a na ph yl ax is ; S PT , s ki n pr ic k te st ; I gE , i m m un og lo bu lin E . Page 23 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 17 380 References 381 1. Sastre J. Molecular diagnosis in allergy. Clin Exp Allergy 2010;40(10):1442-1460. 382 2. Popescu FD. Cross-reactivity between aeroallergens and food allergens. World J 383 Methodol 2015;5(2):31-50. 384 3. Wangorsch A, Larsson H, Messmer M, Garcia-Moral A, Lauer I, Wolfheimer S, et al. 385 Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach 386 associated plane pollen allergy. Clin Exp Allergy 2016;46(5):764-774. 387 4. 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Understanding 488 patient sensitization profiles in complex pollen areas: a molecular epidemiological study. 489 Allergy 2008;63(11):1550-1558. 490 41. Vieths S, Scheurer S, Ballmer-Weber B. Current understanding of cross-reactivity of 491 food allergens and pollen. Ann N Y Acad Sci 2002;964:47-68. 492 42. Perez-Calderon R, Gonzalo-Garijo MA, Rodriguez-Velasco FJ, Sanchez-Vega S, 493 Bartolome-Zavala B. Occupational respiratory allergy in peach crop workers. Allergy 494 2017;72(10):1556-1564. 495 43. Garcia BE, Lombardero M, Echechipia S, Olaguibel JM, Diaz-Perales A, Sanchez- 496 Monge R, et al. Respiratory allergy to peach leaves and lipid-transfer proteins. Clin Exp Allergy 497 2004;34(2):291-295. 498 44. Marzban GM, A.; Herndl, A.; Jäger, S.; Stoyanova, M. E.; Hemmer, W.; Katinger, H.; 499 Laimer, M. Direct evidence for the presence of allergens in Rosaceae fruit tree pollen. 500 Aerobiologia 2006;22(3):8. 501 45. Fernandez-Rivas M, Bolhaar S, Gonzalez-Mancebo E, Asero R, van Leeuwen A, Bohle 502 B, et al. Apple allergy across Europe: how allergen sensitization profiles determine the clinical 503 expression of allergies to plant foods. J Allergy Clin Immunol 2006;118(2):481-488. 504 46. Pascal M, Munoz-Cano R, Reina Z, Palacin A, Vilella R, Picado C, et al. Lipid transfer 505 protein syndrome: clinical pattern, cofactor effect and profile of molecular sensitization to plant- 506 foods and pollens. Clin Exp Allergy 2012;42(10):1529-1539. 507 47. Sanchez-Solares J, Delgado-Dolset MI, Mera-Berriatua L, Hormias-Martin G, 508 Cumplido JA, Saiz V, et al. Respiratory allergies with no associated food allergy disrupt oral 509 mucosa integrity. Allergy 2019. 510 48. Hua X, Goedert JJ, Pu A, Yu G, Shi J. Allergy associations with the adult fecal 511 microbiota: Analysis of the American Gut Project. EBioMedicine 2016;3:172-179. Page 26 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 20 512 Legend to the Figures 513 FIGURE 1. Identification of non-specific lipid transfer proteins cross-reactive to Ole e 7 514 from different pollen and food-derived extracts. ELISA inhibitions were performed with 515 20 or 200 μg of pollen (A) or food (B) protein extracts as inhibitors. A pool of six Ole e 7- 516 positive sera from olive pollen allergic patients sensitized to Ole e 7 was used in the assay. 517 Experiments were performed in duplicate (p<0.05). 518 519 FIGURE 2. Physico-chemical and immunological analysis of recombinant Ole e 7 and Pru 520 p 3. (A) Analysis of 500 ng of the recombinant allergens by Coomassie Blue staining after 521 SDS-PAGE. (B-D) Analysis of common antigenic features of Ole e 7 and Pru p 3 allergens 522 by WB and ELISA. (B) Immunostaining of 500 ng of rOle e 7 and Pru p 3 using the 523 indicated polyclonal antisera (pAb). BSA nitrocellulose blotted was used as negative 524 control of the assay. Signal was developed for 30 seconds using ECL WesternBrightTM 525 QUANTUM (Advansta) reagent for Ole e 7 with anti-Ole e 7 pAb and Pru p 3 with anti- 526 Pru p 3 pAb, and for 3 min for the other determinations. (C-D) Analysis of 100 ng of 527 recombinant allergens coated in ELISA wells with indicated dilutions of Ole e 7- or Pru p 528 3-pAbs. All the experiments were performed in duplicate. 529 530 FIGURE 3. IgE-binding inhibition assays using sera from bisensitized and Ole e 7- 531 monosensitized patients. (A-D) ELISA and WB IgE-binding inhibition assays using sera 532 from a patient bisensitized to Ole e 7 and Pru p 3. (E-F) WB IgE-binding inhibition assays 533 using sera from a patient monosensitized to Ole e 7. ELISA was performed coating 100 ng 534 of recombinant proteins using indicated amounts of inhibitors. WB was performed using 535 200 ng of Pru p 3 or 500 ng of Ole e 7 blotted in nitrocellulose membranes and 2 µg of the 536 indicated inhibitors under denaturing (C-E) or non-denaturing conditions (F). Inhibition 537 values (%) are indicated. Page 27 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 21 538 FIGURE 4. Basophils degranulation (% CD63+) by rOle e 7 and r Pru p 3. (A) 10 µg/mL 539 of rOle e 7, or (B) rPru p 3 in blood samples of the study cohort (n=48). Comparisons 540 between groups of patients of the study cohort (n=48) were performed using U Mann 541 Whitney test. Furthermore, basophil degranulation of the 6 selected patients was analyzed 542 using 0.1 µg/mL, 1 µg/mL and 10 µg/mL of rOle e 7 (C) or rPru p 3 (D). N-Formil-Met- 543 Leu-Phe was used as positive control and phosphate buffer saline as negative control. 544 Mean with standard error of the mean (SEM) bars are displayed. 545 546 FIGURE 5. β-hexosaminidase release assay of Hum RBL-2H3 cells. Hum RBL-2H3 cells 547 were tested at four different concentrations (10-3 µg/mL, 10-2 µg/mL, 10-1 µg/mL, 1 µg/mL) 548 of rOle e 7 or r Pru p 3 using serum of indicated patients to sensitize the cells overnight. 549 Patients 9, 12, 16 and 36 were monosensitized to Ole e 7. Patients 37 and 38 showed 550 bisensitization to Ole e 7 and Pru p 3. An absence of β-hexosaminidase release was 551 observed without allergen stimulation (negative control). Page 28 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 0 20 40 60 80 100 120 Ig E -b in d in g I n h ib it io n ( % ) Food extracts 20µg 200µg Figure 1 A B 0 20 40 60 80 100 120 Ig E -b in d in g I n h ib it io n ( % ) Pollen extracts 20 µg 200 µg Page 29 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 2 A B C D 17 27 33 46 79 102 10 kDa Ole e 7 pAb Pru p 3 pAb 3 2.5 2 0.5 S p e c i f i c I g G l e v e l s ( O D a t 4 9 2 n m ) Dilution Factor 0 Ole e 7 pAb 3 2.5 2 0.5 Dilution Factor 0 Pru p 3 pAb Page 30 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 3 A C B D 17 kDa 17 kDa 17 27 33 46 79 102 kDa 17 27 33 46 79 102 kDa rOle e 7rOle e 7 100 100- 100 25- Patient 28 Patient 30 100 15- Patient 28 E F B is en si tiz ed to O le e 7 a nd P ru p 3 (P at ie nt 44 ) M on os en si tiz ed to O le e 7 Inhibition (%) Inhibition (%)- 100 100 - 25 100 rOle e 7-coated wells rPru p 3-coated wells Inhibitors (µg) rOle e 7 rPru p 3 Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Page 31 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 4 rPru p 3 A B C D O le e 7 % b a s o p h il s M o n o s e n s itiz e d to O le e 7 M o n o s e n s itiz e d to P ru p 3 B is e n s itiz e d 0 2 0 4 0 6 0 8 0 1 0 0 P ru p 3 % b a s o p h il s M o n o s e n s itiz e d to O le e 7 M o n o s e n s itiz e d to P ru P 3 B is e n s itiz e d 0 2 0 4 0 6 0 8 0 1 0 0 % C D 6 3 0 .1 µ g /m L 1 µ g /m L 1 0 µ g /m L 0 2 0 4 0 6 0 8 0 1 0 0 P9 P1 2 P1 6 P3 6 P3 7 P3 8 B a s o p h il s ( % ) Patient sensitization Patient sensitization Ole e 7 Pru p 3 Ole e 7 + Pru p 3 Ole e 7 Pru p 3 Ole e 7 + Pru p 3 p=0.864 p=0.078 p=0.001 p<0.001 p=0.699 P=0.191 rOle e 7 rPru p 3 0.1 µg/mL 1 µg/mL 10 µg/mL % C D 6 3 0 .1 µ g /m L 1 µ g /m L 1 0 µ g /m L 0 2 0 4 0 6 0 8 0 1 0 0 P9 P1 2 P1 6 P3 6 P3 7 P3 8 % C D 6 3 0 .1 µ g /m L 1 µ g /m L 1 0 µ g /m L 0 2 0 4 0 6 0 8 0 1 0 0 P9 P1 2 P1 6 P3 6 P3 7 P3 8 rOle e 7 0.1 µg/mL 1 µg/mL 10 µg/mL P16 38 P12 P37 9 P36 P38 P16 P9 P37 P36 P12 Page 32 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 5 β -H e x o s a m in id a s e ( % ) β -H e x o s a m in id a s e ( % ) 0 10 20 30 40 50 0 0.001 0.01 0.1 1 Patient 9 0 10 20 30 40 50 0 0.001 0.01 0.1 1 0 10 20 30 40 50 0 0.001 0.01 0.1 1 Patient 38 Patient 37 0 10 20 30 40 50 0 0.001 0.01 0.1 1 Patient 36 0 10 20 30 40 50 0 0.001 0.01 0.1 1 Patient 16 0 10 20 30 40 50 0 0.001 0.01 0.1 1 Patient 12 . . . . . . rOle e 7 rPru p 3 Protein (µg/µL) Protein (µg/µL) Protein (µg/µL) Protein (µg/µL) Protein (µg/µL) Protein (µg/µL) Page 33 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 1 Supporting Information 2 3 New insights into the sensitization to non-related nsLTPs from pollen and food 4 5 Carmen Oeo-Santos, BSa*, Ana Navas, BS b, c*, Sara Benedé, PhDa, Berta Ruíz-León, MD, 6 PhD b, c,d, Araceli Díaz-Perales, PhDe,d, Lothar Vogel, PhDe, Carmen Moreno-Aguilar, MD, 7 PhD b, c,d, Aurora Jurado, MD, PhD b, c,d, Mayte Villalba, PhD a,d,^, Rodrigo Barderas, 8 PhDf,^. 9 10 * Both authors contributed equally. 11 12 a Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 13 Universidad Complutense de Madrid, 28040 Madrid, Spain. 14 b UGC Inmunología y Alergia, Hospital Universitario Reina Sofía de Córdoba, 14004 Córdoba, 15 Spain. 16 c Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/ Hospital 17 Universitario Reina Sofía/ Universidad de Córdoba, 14004 Córdoba, Spain. 18 d Allergy Network ARADyAL. Instituto de Salud Carlos III, Madrid, Spain. 19 e Centro de Biotecnología y Genómica de Plantas (CBGP, UPM-INIA), Campus de 20 Montegancedo-UPM, 28223 Madrid, Spain. 21 f Division of Allergology, Paul-Erlich-Institut, 63225, Langen, Germany. 22 g UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, Majadahonda 28220, 23 Madrid, Spain. 24 25 ^ To whom correspondence should be addressed: 26 Rodrigo Barderas. 27 Functional Proteomics Unit, UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, 28 E-28222 Majadahonda, Madrid, Spain; Tel.: 34-91-8223231; E-mail: r.barderasm@isciii.es 29 30 ^ To whom correspondence should be addressed: 31 Mayte Villalba. 32 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 33 Universidad Complutense de Madrid, E-28040 Madrid, Spain; Tel.: 34-91-3944155; E-mail: 34 mvillalb@ucm.es Page 34 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 35 Materials and Methods 36 37 Protein extracts, allergens and antibodies 38 Olive pollen was obtained from ALK-Abelló (Madrid, Spain). Pollens from other 39 biological sources were obtained from Allergon (1). Pollen protein extracts were prepared by 40 saline extraction. To prepare food-derived extracts, plant foods were crushed under liquid 41 nitrogen to obtain fine flour. Proteins were extracted by homogenization in 0.2 M ammonium 42 bicarbonate buffer, pH 8.0, containing 1 mM PMSF and stirred for one hour at 4ºC. 43 Delipidation was performed using cold acetone. Protein extract concentration was determined 44 according to Lowry et al. (2). 45 Ole e 7 and Pru p 3 expression and purification were performed according to established 46 protocols (3, 4). Purity of both allergens was confirmed by mass spectrometry on an Autoflex 47 III MALDI-TOF-TOF instrument (Bruker Daltonics, Bremen, Germany), respectively 48 (Supporting Figure 2). 49 Ole e 7 and Pru p 3 rabbit polyclonal antisera (pAb) were previously obtained 50 accomplishing the ethics guidelines of the Complutense University of Madrid and Fundación 51 Jiménez Díaz, respectively (3-6). Horseradish peroxidase-labeled goat polyclonal antibody 52 against rabbit IgG was purchased from BioRad Laboratories Inc (Richmond, CA, USA). 53 Horseradish peroxidase-labeled mouse anti-human IgE was purchased from Southern Biotech. 54 55 IgG-binding analysis 56 The IgG binding of Ole e 7- and Pru p 3-specific polyclonal antisera to Ole e 7 and Pru 57 p 3 allergens was analyzed by ELISA and WB. Indirect ELISA was performed in duplicate in 58 96-well plates (CorningTM) coated with 0.1 µg of the indicated proteins. A titration of the 59 specific polyclonal antisera to Ole e 7 and Pru p 3 was performed with concentrations ranging 60 from 10-2 to 10-6 µg/µL. Alternatively, a goat anti-rabbit IgG horseradish peroxidase-labeled 61 antibody (1:3000) or a goat anti-mouse IgG horseradish peroxidase-labeled antibody (1:2500) 62 was used to determine the IgG recognition to each allergen. For WB analysis, proteins were Page 35 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 63 transferred to nitrocellulose membrane after SDS-PAGE under reducing conditions (2- 64 mercaptoethanol 5%). Immunodetection was achieved by incubating with specific polyclonal 65 antisera to Ole e 7 and Pru p 3 (1:10000, 1:1000, respectively) and an anti-rabbit IgG 66 horseradish peroxidase-labeled polyclonal antibody (1:3000). Chemiluminescent signal was 67 developed using ECL-Western blotting reagent (Amersham Bioscience) or WesternBrightTM 68 QUANTUM (Advansta) reagents. 69 70 Specific IgE binding to Ole e 7 and Pru p 3 71 Specific IgE levels were determined by ImmunoCAP 250 (Thermo Scientific, Uppsala, 72 Sweden) and indirect ELISA. Values >0.35 kU/L and OD at 492 nm >0.1 by ImmunoCAP 250 73 and ELISA, respectively, were considered positive. Indirect ELISA was performed in duplicate 74 in 96-well plates (CorningTM) coated with 0.1 µg of recombinant protein per well, according to 75 previously optimized protocols (1, 3, 7). Binding of human IgE was detected by a horseradish 76 peroxidase-labeled mouse anti-human IgE Fc (1:1000) (Southern Biotech). Peroxidase reaction 77 was detected by using 50 μL per well of 0.63 mg/mL o-Phenylenediamine in 0.1 M sodium 78 citrate 4% methanol containing 1.6 μL/mL 30% H2O2. The reaction was stopped with 50 μL 3N 79 H2SO4. Signal was measured at 492 nm in an iMark microplate absorbance reader (Bio-Rad). 80 81 Cell-based allergic mediator release assays 82 Evaluation of specific cell mediators of allergic response against Ole e 7 and Pru p 3 83 was determined by Basophil Activation Test (BAT) and RBL-2H3 activation cells. BAT was 84 performed using rOle e 7 and rPru p 3 allergens obtained as described above. Briefly, 100 µL of 85 heparin anticoagulated total blood from each allergic patient were incubated with Basophil 86 Stimulation Buffer (ref. 339664, BD Biosciences, CA, USA) for 10 minutes at 37 ºC. Then, 100 87 µL of phosphate buffer saline (PBS) was added as negative control and 50 µL of N-Formyl- 88 Met-Leu-Phe (f-MLP) at 2 µM concentration as positive control. Either rOle e 7 or rPru p 3 89 allergens were also added at 0.1 µg/mL, 1 µg/mL and 10 µg/mL in separate tubes. All tubes 90 were incubated for 30 minutes at 37 ºC. Basophil degranulation was stopped by transferring Page 36 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 91 sample tubes to an ice bath for 5 minutes. Cell staining was performed using CD63- 92 FITC/CD123-PE/anti-HLA-DR-PerCP cocktail (ref. 341068, BD FastImmuneTM, Becton, 93 Dickinson and Company, San Jose, CA, USA). After lysing cells with 2 mL of 1x BD FACSTM 94 lysing solution and washing twice with PBS, stained cells were acquired in a BD FacsCanto II 95 cytometer (Becton Dickinson and Company, San Jose, CA, USA) using BD FacsDivaTM as 96 acquisition and analysis software. The specificity of the response to rOle e 7 and rPru p 3 was 97 ascertained using a cohort of non-allergic donors. 98 RBL-2H3 cell experiments were developed as previously described (3), with slight 99 modifications. Briefly, cells were sensitized overnight at 37ºC with 5 or 10% of sera from 100 allergic patients. After 12 h, cells were stimulated with either rOle e 7 or rPru p 3 at four 101 different concentrations (10-3 µg/mL, 10-2 µg/mL, 10-1 µg/mL and 1 µg/mL). 102 103 Inhibition assays 104 Inhibition assays were performed by ELISA and WB. For ELISA inhibition tests, 0.1 105 µg of purified proteins were coated overnight at 4ºC to CorningTM 96 well plates. Samples were 106 coated in duplicates. Individual human sera (diluted 1:10) or an equivolumetric pool of human 107 sera (n=6), previously incubated with 0.2 µg or 2 µg of the specific recombinant protein, or with 108 20 µg or 200 µg of pollen or food-derived extracts as inhibitors. IgE-binding was detected by a 109 horseradish peroxidase-labeled mouse anti-human IgE Fc (1:1000) (Southern Biotech). 110 For WB inhibition assays, allergenic proteins were alternatively transferred under 111 denaturing and non-denaturing conditions to analyze the relevance of epitope conformation in 112 IgE-binding. Hence, under denaturing conditions, proteins showed mostly linear epitopes, while 113 under non-denaturing conditions binding to conformational epitopes may be determined. 114 Immunodetection on membranes was achieved by incubating with individual human sera (1:10), 115 previously incubated with 2 µg of inhibitor, and a horseradish peroxidase-labeled mouse anti- 116 human IgE Fc (1:1000). Chemiluminescent signal was developed using ECL-Western blotting 117 reagent (Amersham Bioscience) or WesternBrightTM QUANTUM (Advansta) reagents. Page 37 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 118 References 119 1. Mas S, Oeo-Santos C, Cuesta-Herranz J, Diaz-Perales A, Colas C, Fernandez J, et al. A 120 relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is 121 a natural degradation product of an integral 47kDa polygalaturonase. Biochim Biophys Acta 122 2017;1865(8):1067-1076. 123 2. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin 124 phenol reagent. J Biol Chem 1951;193(1):265-275. 125 3. Oeo-Santos C, Mas S, Benede S, Lopez-Lucendo M, Quiralte J, Blanca M, et al. A 126 recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass 127 spectrometry retains the allergenic ability of the natural allergen. J Proteomics 2018;187:39-46. 128 4. Diaz-Perales A, Garcia-Casado G, Sanchez-Monge R, Garcia-Selles FJ, Barber D, 129 Salcedo G. cDNA cloning and heterologous expression of the major allergens from peach and 130 apple belonging to the lipid-transfer protein family. Clin Exp Allergy 2002;32(1):87-92. 131 5. Diaz-Perales A, Lombardero M, Sanchez-Monge R, Garcia-Selles FJ, Pernas M, 132 Fernandez-Rivas M, et al. Lipid-transfer proteins as potential plant panallergens: cross- 133 reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different 134 IgE-binding capacities. Clin Exp Allergy 2000;30(10):1403-1410. 135 6. Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and 136 characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 137 1999;104(4 Pt 1):797-802. 138 7. Oeo-Santos C, Mas S, Quiralte J, Colas C, Blanca M, Fernandez J, et al. A 139 hypoallergenic polygalacturonase isoform from olive pollen is implicated in pollen-pollen 140 cross-reactivity. Int Arch Allergy Immunol 2018:1-12. Page 38 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Supporting Fig 1. Comparison of the amino acid sequence of Ole e 7 with Pru p 3 and Pyr c 3. Identity and Similarity percentage values among indicated allergenic nsLTPs are shown. Dialign was used to align the amino acid sequences of the proteins. Genedoc was used for visualization and calculation of Identity and Similarity percentages. Ole e 7 100 31 30 100 51 46 Ole e 7 Pru p 3 Pyr c 3 Ole e 7 Pru p 3 Pyr c 3 Identity (%) Similarity (%) Page 39 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 1 Supporting Information 2 3 New insights into the sensitization to non-related nsLTPs from pollen and food 4 5 Carmen Oeo-Santos, BSa*, Ana Navas, BS b, c*, Sara Benedé, PhDa, Berta Ruíz-León, MD, 6 PhD b, c,d, Araceli Díaz-Perales, PhDe,d, Lothar Vogel, PhDe, Carmen Moreno-Aguilar, MD, 7 PhD b, c,d, Aurora Jurado, MD, PhD b, c,d, Mayte Villalba, PhD a,d,^, Rodrigo Barderas, 8 PhDf,^. 9 10 * Both authors contributed equally. 11 12 a Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 13 Universidad Complutense de Madrid, 28040 Madrid, Spain. 14 b UGC Inmunología y Alergia, Hospital Universitario Reina Sofía de Córdoba, 14004 Córdoba, 15 Spain. 16 c Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/ Hospital 17 Universitario Reina Sofía/ Universidad de Córdoba, 14004 Córdoba, Spain. 18 d Allergy Network ARADyAL. Instituto de Salud Carlos III, Madrid, Spain. 19 e Centro de Biotecnología y Genómica de Plantas (CBGP, UPM-INIA), Campus de 20 Montegancedo-UPM, 28223 Madrid, Spain. 21 f Division of Allergology, Paul-Erlich-Institut, 63225, Langen, Germany. 22 g UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, Majadahonda 28220, 23 Madrid, Spain. 24 25 ^ To whom correspondence should be addressed: 26 Rodrigo Barderas. 27 Functional Proteomics Unit, UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, 28 E-28222 Majadahonda, Madrid, Spain; Tel.: 34-91-8223231; E-mail: r.barderasm@isciii.es 29 30 ^ To whom correspondence should be addressed: 31 Mayte Villalba. 32 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, 33 Universidad Complutense de Madrid, E-28040 Madrid, Spain; Tel.: 34-91-3944155; E-mail: 34 mvillalb@ucm.es Page 66 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 35 Materials and Methods 36 37 Protein extracts, allergens and antibodies 38 Olive pollen was obtained from ALK-Abelló (Madrid, Spain). Pollens from other 39 biological sources were obtained from Allergon (1). Pollen protein extracts were prepared by 40 saline extraction. To prepare food-derived extracts, plant foods were crushed under liquid 41 nitrogen to obtain fine flour. Proteins were extracted by homogenization in 0.2 M ammonium 42 bicarbonate buffer, pH 8.0, containing 1 mM PMSF and stirred for one hour at 4ºC. 43 Delipidation was performed using cold acetone. Protein extract concentration was determined 44 according to Lowry et al. (2). 45 Ole e 7 and Pru p 3 expression and purification were performed according to established 46 protocols (3, 4). Purity of both allergens was confirmed by mass spectrometry on an Autoflex 47 III MALDI-TOF-TOF instrument (Bruker Daltonics, Bremen, Germany), respectively 48 (Supporting Figure 2). 49 Ole e 7 and Pru p 3 rabbit polyclonal antisera (pAb) were previously obtained 50 accomplishing the ethics guidelines of the Complutense University of Madrid and Fundación 51 Jiménez Díaz, respectively (3-6). Horseradish peroxidase-labeled goat polyclonal antibody 52 against rabbit IgG was purchased from BioRad Laboratories Inc (Richmond, CA, USA). 53 Horseradish peroxidase-labeled mouse anti-human IgE was purchased from Southern Biotech. 54 55 IgG-binding analysis 56 The IgG binding of Ole e 7- and Pru p 3-specific polyclonal antisera to Ole e 7 and Pru 57 p 3 allergens was analyzed by ELISA and WB. Indirect ELISA was performed in duplicate in 58 96-well plates (CorningTM) coated with 0.1 µg of the indicated proteins. A titration of the 59 specific polyclonal antisera to Ole e 7 and Pru p 3 was performed with concentrations ranging 60 from 10-2 to 10-6 µg/µL. Alternatively, a goat anti-rabbit IgG horseradish peroxidase-labeled 61 antibody (1:3000) or a goat anti-mouse IgG horseradish peroxidase-labeled antibody (1:2500) 62 was used to determine the IgG recognition to each allergen. For WB analysis, proteins were Page 67 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 63 transferred to nitrocellulose membrane after SDS-PAGE under reducing conditions (2- 64 mercaptoethanol 5%). Immunodetection was achieved by incubating with specific polyclonal 65 antisera to Ole e 7 and Pru p 3 (1:10000, 1:1000, respectively) and an anti-rabbit IgG 66 horseradish peroxidase-labeled polyclonal antibody (1:3000). Chemiluminescent signal was 67 developed using ECL-Western blotting reagent (Amersham Bioscience) or WesternBrightTM 68 QUANTUM (Advansta) reagents. 69 70 Specific IgE binding to Ole e 7 and Pru p 3 71 Specific IgE levels were determined by ImmunoCAP 250 (Thermo Scientific, Uppsala, 72 Sweden) and indirect ELISA. Values >0.35 kU/L and OD at 492 nm >0.1 by ImmunoCAP 250 73 and ELISA, respectively, were considered positive. Indirect ELISA was performed in duplicate 74 in 96-well plates (CorningTM) coated with 0.1 µg of recombinant protein per well, according to 75 previously optimized protocols (1, 3, 7). Binding of human IgE was detected by a horseradish 76 peroxidase-labeled mouse anti-human IgE Fc (1:1000) (Southern Biotech). Peroxidase reaction 77 was detected by using 50 μL per well of 0.63 mg/mL o-Phenylenediamine in 0.1 M sodium 78 citrate 4% methanol containing 1.6 μL/mL 30% H2O2. The reaction was stopped with 50 μL 3N 79 H2SO4. Signal was measured at 492 nm in an iMark microplate absorbance reader (Bio-Rad). 80 81 Cell-based allergic mediator release assays 82 Evaluation of specific cell mediators of allergic response against Ole e 7 and Pru p 3 83 was determined by Basophil Activation Test (BAT) and RBL-2H3 activation cells. BAT was 84 performed using rOle e 7 and rPru p 3 allergens obtained as described above. Briefly, 100 µL of 85 heparin anticoagulated total blood from each allergic patient were incubated with Basophil 86 Stimulation Buffer (ref. 339664, BD Biosciences, CA, USA) for 10 minutes at 37 ºC. Then, 100 87 µL of phosphate buffer saline (PBS) was added as negative control and 50 µL of N-Formyl- 88 Met-Leu-Phe (f-MLP) at 2 µM concentration as positive control. Either rOle e 7 or rPru p 3 89 allergens were also added at 0.1 µg/mL, 1 µg/mL and 10 µg/mL in separate tubes. All tubes 90 were incubated for 30 minutes at 37 ºC. Basophil degranulation was stopped by transferring Page 68 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 91 sample tubes to an ice bath for 5 minutes. Cell staining was performed using CD63- 92 FITC/CD123-PE/anti-HLA-DR-PerCP cocktail (ref. 341068, BD FastImmuneTM, Becton, 93 Dickinson and Company, San Jose, CA, USA). After lysing cells with 2 mL of 1x BD FACSTM 94 lysing solution and washing twice with PBS, stained cells were acquired in a BD FacsCanto II 95 cytometer (Becton Dickinson and Company, San Jose, CA, USA) using BD FacsDivaTM as 96 acquisition and analysis software. The specificity of the response to rOle e 7 and rPru p 3 was 97 ascertained using a cohort of non-allergic donors. 98 RBL-2H3 cell experiments were developed as previously described (3), with slight 99 modifications. Briefly, cells were sensitized overnight at 37ºC with 5 or 10% of sera from 100 allergic patients. After 12 h, cells were stimulated with either rOle e 7 or rPru p 3 at four 101 different concentrations (10-3 µg/mL, 10-2 µg/mL, 10-1 µg/mL and 1 µg/mL). 102 103 Inhibition assays 104 Inhibition assays were performed by ELISA and WB. For ELISA inhibition tests, 0.1 105 µg of purified proteins were coated overnight at 4ºC to CorningTM 96 well plates. Samples were 106 coated in duplicates. Individual human sera (diluted 1:10) or an equivolumetric pool of human 107 sera (n=6), previously incubated with 0.2 µg or 2 µg of the specific recombinant protein, or with 108 20 µg or 200 µg of pollen or food-derived extracts as inhibitors. IgE-binding was detected by a 109 horseradish peroxidase-labeled mouse anti-human IgE Fc (1:1000) (Southern Biotech). 110 For WB inhibition assays, allergenic proteins were alternatively transferred under 111 denaturing and non-denaturing conditions to analyze the relevance of epitope conformation in 112 IgE-binding. Hence, under denaturing conditions, proteins showed mostly linear epitopes, while 113 under non-denaturing conditions binding to conformational epitopes may be determined. 114 Immunodetection on membranes was achieved by incubating with individual human sera (1:10), 115 previously incubated with 2 µg of inhibitor, and a horseradish peroxidase-labeled mouse anti- 116 human IgE Fc (1:1000). Chemiluminescent signal was developed using ECL-Western blotting 117 reagent (Amersham Bioscience) or WesternBrightTM QUANTUM (Advansta) reagents. Page 69 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 118 References 119 1. Mas S, Oeo-Santos C, Cuesta-Herranz J, Diaz-Perales A, Colas C, Fernandez J, et al. A 120 relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is 121 a natural degradation product of an integral 47kDa polygalaturonase. Biochim Biophys Acta 122 2017;1865(8):1067-1076. 123 2. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin 124 phenol reagent. J Biol Chem 1951;193(1):265-275. 125 3. Oeo-Santos C, Mas S, Benede S, Lopez-Lucendo M, Quiralte J, Blanca M, et al. A 126 recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass 127 spectrometry retains the allergenic ability of the natural allergen. J Proteomics 2018;187:39-46. 128 4. Diaz-Perales A, Garcia-Casado G, Sanchez-Monge R, Garcia-Selles FJ, Barber D, 129 Salcedo G. cDNA cloning and heterologous expression of the major allergens from peach and 130 apple belonging to the lipid-transfer protein family. Clin Exp Allergy 2002;32(1):87-92. 131 5. Diaz-Perales A, Lombardero M, Sanchez-Monge R, Garcia-Selles FJ, Pernas M, 132 Fernandez-Rivas M, et al. Lipid-transfer proteins as potential plant panallergens: cross- 133 reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different 134 IgE-binding capacities. Clin Exp Allergy 2000;30(10):1403-1410. 135 6. Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and 136 characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 137 1999;104(4 Pt 1):797-802. 138 7. Oeo-Santos C, Mas S, Quiralte J, Colas C, Blanca M, Fernandez J, et al. A 139 hypoallergenic polygalacturonase isoform from olive pollen is implicated in pollen-pollen 140 cross-reactivity. Int Arch Allergy Immunol 2018:1-12. Page 70 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Supporting Fig 2. Quality control of the recombinant proteins of the study after expression and purification. Purity of rOle e 7 and rPru p 3 was confirmed by mass spectrometry (MALDI-TOF-TOF). 3962.1 9812.5 3235.0 4521.8 0 1000 2000 3000 4000 In te n s . [a .u .] 5000 10000 15000 20000 25000 m/z In te n s it y [ a .u .] 9812.5 10000 15000 8000 10000 m/z 10000 8000 6000 4000 2000 B In te n s it y [ a .u .] 0 10000 8000 6000 4000 2000 0 rOle e 7 399 9 .0 76 1 4 .4 1 1 2 29 .8 148 45 .2 184 60 .6 220 76 .0 M ass (m /z ) 2 .1E+4 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 % I n t e n s i t y TOF/TOF™ Linear S pec #1 MC=>S M7[BP = 9270.9, 20682] 9 2 6 4 .2 1 3 9 9 4 2 9 .6 6 0 2 9 5 9 1 .9 2 5 8 4 6 3 5 .0 2 6 4 9 7 5 3 .4 2 7 7 1 8 5 2 9 .3 6 1 3 8 8 3 8 .5 7 6 24 7 1 5 .2 1 9 7 5 6 9 7 .5 0 4 9 A m/z 3962.1 9812.5 3235.0 4521.8 0 1000 2000 3000 4000 In te n s . [a .u .] 5000 10000 15000 20000 25000 m/z 399 9 .0 761 4 .4 1 12 29 .8 1 48 45 .2 1 84 60 .6 22 0 76 .0 M as s (m /z ) 2 .1 E+4 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 % I n t e n s i t y TOF/TOF™ Linear S pec #1 MC=>S M7[BP = 9270.9, 20682] 9 2 6 4 .2 1 3 9 9 4 2 9 .6 6 0 2 9 5 9 1 .9 2 5 8 4 6 3 5 .0 2 6 4 9 7 5 3 .4 2 7 7 1 8 5 2 9 .3 6 1 3 8 8 3 8 .5 7 6 24 7 1 5 .2 1 9 7 5 6 9 7 .5 0 4 9 399 9 .0 761 4 .4 112 29 .8 148 45 .2 184 60 .6 220 76 .0 M ass (m /z ) 2 .1E+4 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 % I n t e n s i t y TOF/TOF™ Linear S pec #1 MC=>S M7[BP = 9270.9, 20682] 9 2 6 4 .2 1 3 9 9 4 2 9 .6 6 0 2 9 5 9 1 .9 2 5 8 4 6 3 5 .0 2 6 4 9 7 5 3 .4 2 7 7 1 8 5 2 9 .3 6 1 3 8 8 3 8 .5 7 6 24 7 1 5 .2 1 9 7 5 6 9 7 .5 0 4 9 9264.2 rPru p 3 429.7 9591.9 8838.6 3962.1 9812.5 3235.0 4521.8 0 1000 2000 3000 4000 In te n s . [a .u .] 5000 10000 15000 20000 25000 m/z Page 40 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Supporting Fig 3. Detection of specific IgE levels from serum of patients to Ole e 7 and Pru p 3 by ImmunoCAP (A) and ELISA (B).Values above 0.1 OD by ELISA and 0.35 kUA/L by ImmunoCAP were considered positive. Right, Mean ± standard error of the mean (SEM) of the IgE binding to Ole e 7 (natural or recombinant protein for ImmunoCAP and ELISA, respectively) and rPru p 3 for all positive serum from Córdoba are also represented. ELISA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 0,0 0,5 1,0 1,5 3,0 3,5 rOle e 7 rPru p 3 ImmunoCAP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 0 100 200 500 600 rOle e 7 rPru p 3 A B S pe ci fic Ig E le ve ls (k U /L ) S pe ci fic Ig E le ve ls (O D a t 4 9 2 n m ) Sera number Sera number . . . . . . 80 60 40 20 0 S p e c if ic I g E l e v e ls ( k U /L ) 1 0.5 0 S p e c if ic I g E l e v e ls ( O D a t 4 9 2 n m ) Page 41 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Supporting Fig 4. IgE-binding inhibition assay by ELISA from bisensitized patients to Ole e 7 and Pru p 3. Inhibition of the IgE-binding to 100 ng of rOle e 7 or rPru p 3 was determined by ELISA in comparison to the IgE-binding signal without inhibitor. Amounts of inhibitor (µg) are also indicated in the figure. 0 20 40 60 80 100 Patient 1 Patient 21 Patient 27 Ig E -b in d in g i n h ib it io n ( % ) rOle e 7-coated wells 0.02 µg Ole e 7 2 µg Ole e 7 0.02 µg Pru p 3 2 µg Pru p 3 0 20 40 60 80 100 Patient 1 Patient 21 Patient 27 Ig E -b in d in g i n h ib it io n ( % ) rPru p 3-coated wells 0.02 µg Ole e 7 2 µg Ole e 7 0.02 µg Pru p 3 2 µg Pru p 3 Page 42 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Negative control Positive control CD123 HLA-DR CD63 CD63 S S C C D 1 2 3 C D 1 2 3 C D 1 2 3 Supporting Fig 5. Gating strategy. SSC, side scatter. Basophils were identified according to the expression of CD123 and HLA-DR markers (CD123+HLA-DR-). Degranulated basophils were identified according to the expression of CD63 marker (CD63+). Results from healthy individual blood cells stimulated with phosphate buffer saline (negative control) and N-Formil-Met-Leu-Phe (positive control) are shown. Page 43 of 70 Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Monosensitized to Ole e 7 (n=13) Monosensitized to Pru p 3 (n=7) Bisensitized (n=28) Age, mean (SD) 36.7 (10.14) 42.14 (14.49) 31.46 (10.27) Female, (%) 46.15 85.71 60.71 Habitat Rural (%) 46.15 14.29 50 Urban (%) 53.85 85.71 50 Respiratory clinical symptoms Rhinitis (%) 15.38 28.57 21.43 Asthma (%) 0 14.29 7.14 Both (%) 84.62 42.86 64.29 Clinical symptoms after Rosaceae consumption Cutaneous (%) 0 28.57 14.29 Cutaneous and OAS (%) 0 0 14.29 OAS (%) 0 42.86 14.29 Anaphylaxis (%) 0 28.57 42.86 No symptoms (%) 0 0 7.14 Positive SPT to Olea europaea (%) 100 0 92.86 Positive SPT to Pru p 3 (%) 0 71.43 60.71 IgE to Ole e 7 by ImmunoCAP, mean (SD) 62.37 (42.50) 0.11 (0.08) 97.39 (142.11) IgE to Pru p 3 by ImmunoCAP, mean (SD) 0.08 (0.12) 23.65 (50.51) 12.92 (31.84) IgE to rOle e 7 by ELISA, mean (SD) 0.49 (0.35) 0.06 (0.13) 0.71 (0.97) IgE to rPru p 3 by ELISA, mean (SD) 0 (0) 0.22 (0.30) 0.25 (0.45) Supporting Table 1 Supporting Table 1. Clinical characteristics of all recruited patients to whom Basophil Activation Test (BAT) was performed. OAS, oral allergy syndrome; IgE, immunoglobulin E. IgE values are indicated in kU/L for ImmunoCAP or in DO (arbitrary units). Page 44 of 70Allergy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60