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patients are allergic to the delivered protein. Hence, if a large amount of allergen penetrates beyond the nonvascularized epidermis toward the dermal vasculature, systemic allergic reactions are frequent, as observed in 6 patients after abrasion. In contrast, only 1 patient developed a systemic allergic reaction after tape-stripping, which has been shown to primarily affect the stratum corneum, with allergen deposition in epidermal layers.7 Eczema was reported after allergen EPIT with abrasion and with tape-stripping but in no patient after placebo EPIT. Please note patient self-reporting of eczema as a weakness of this study. The frequency of adverse effects after allergen EPIT with tapestripping is comparable to our previous experience5,6 (see Table E2) except for 2 striking differences: (1) in the first study with 48-hour patch administration time, more eczema reactions were reported, and (2) in the present subanalysis, the number of systemic adverse effects was more than doubled when using abrasion than when using tape-stripping. As a limitation, we could not show statistical significance for difference in systemic allergic reaction frequency after allergen EPIT with abrasion and tape-stripping when strictly using data of the present subanalysis of ClinicalTrials.gov NCT00777374. Because end points were efficacy and immunological changes, no power calculations were done for the present retrospective analysis. As another limitation, skin abrasion using a foot-file is difficult to standardize and the procedure was performed by different study team members. Furthermore, because abrasion had to be stopped prematurely for safety reasons, no analysis with respect to enhanced efficacy could be performed. Also, it would be interesting to measure the modulation of immune responses after abrasion versus tape-stripping because the degree of skin disruption has been suggested to play a role in polarizing TH1, TH2, or T-regulatory-cell–type responses by activating different subsets of skin-resident antigen-presenting cells.8 Skin-barrier disruption has been reported to favor TH2-polarized immune responses,1 while hydration-facilitated antigen delivery on nondisrupted skin favors T-regulatory-cell responses.9 Hence, skin-barrier disruption would seem disadvantageous for allergen immunotherapy. Nevertheless, we previously showed the therapeutic efficacy of allergen EPIT with tape-stripping.5,6 Therefore, the effects of different methods for skin disruption on T-cell polarization are so far unclear. Here, we highlight that different skin-disruption methods must also be compared for safety, adding another layer of complexity in the field of allergy immunotherapy. Seraina von Moos, MDa P
al Johansen, PhDb Fabian Tay, MDc Nicole Graf, PhDd Thomas M. K€ undig, MDb Gabriela Senti, MDa From the Departments of aInternal Medicine and bDermatology, University Hospital Zurich, and cthe Clinical Trials Center, University of Zurich, University Hospital Zurich, Zurich, Switzerland; and dGraf Biostatistics, Winterthur, Switzerland. E-mail: [email protected]. This study was supported by the Swiss National Science Foundation (grant no. 320030_140902). Disclosure of potential conflict of interest: T. M. K€undig has received research support from the Swiss National Science Foundation (Commission for Innovation and Technology, EU-FP7 grant 32003B_124703); has received consultancy fees from NeoVacs SA, FIO Partners, and AC Immune; and is named as an inventor on patents (belonging to the University of Zurich) on intralymphatic immunotherapy and epicutaneous immunotherapy. The rest of the authors declare that they have no relevant conflicts of interest.

REFERENCES 1. De Benedetto A, Kubo A, Beck LA. Skin barrier disruption: a requirement for allergen sensitization? J Invest Dermatol 2012;132:949-63. 2. Frerichs DM, Ellingsworth LR, Frech SA, Flyer DC, Villar CP, Yu J, et al. Controlled, single-step, stratum corneum disruption as a pretreatment for immunization via a patch. Vaccine 2008;26:2782-7. 3. Glenn GM, Villar CP, Flyer DC, Bourgeois AL, McKenzie R, Lavker RM, et al. Safety and immunogenicity of an enterotoxigenic Escherichia coli vaccine patch containing heat-labile toxin: use of skin pretreatment to disrupt the stratum corneum. Infect Immun 2007;75:2163-70. 4. Nickoloff BJ, Naidu Y. Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. J Am Acad Dermatol 1994;30: 535-46. 5. Senti G, von Moos S, Tay F, Graf N, Sonderegger T, Johansen P, et al. Epicutaneous allergen-specific immunotherapy ameliorates grass pollen-induced rhinoconjunctivitis: a double-blind, placebo-controlled dose escalation study. J Allergy Clin Immunol 2012;129:128-35. 6. Senti G, Graf N, Haug S, Ruedi N, von Moos S, Sonderegger T, et al. Epicutaneous allergen administration as a novel method of allergen-specific immunotherapy. J Allergy Clin Immunol 2009;124:997-1002. 7. Chen X, Shah D, Kositratna G, Manstein D, Anderson RR, Wu MX. Facilitation of transcutaneous drug delivery and vaccine immunization by a safe laser technology. J Control Release 2012;159:43-51. 8. Merad M, Ginhoux F, Collin M. Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nat Rev Immunol 2008;8: 935-47. 9. Dioszeghy V, Mondoulet L, Dhelft V, Ligouis M, Puteaux E, Benhamou PH, et al. Epicutaneous immunotherapy results in rapid allergen uptake by dendritic cells through intact skin and downregulates the allergen-specific response in sensitized mice. J Immunol 2011;186:5629-37. http://dx.doi.org/10.1016/j.jaci.2014.07.037

IgE antibodies to mountain cedar pollen predominantly recognize multiple conformational epitopes on Jun a 1 To the Editor: Recent analyses of several databases of allergens provide evidence for structural similarities of allergenic proteins.1 The 707 allergens with known sequences belong to only 184 (;2%) of the 9318 protein families (Pfams).2 Furthermore, of the rare Pfams that contain an allergen, 81 contain multiple allergens and 10 Pfams with the most allergens contain 300 (42%) allergens. The congruence of pollen allergens with structural families is even more apparent. Of the 157 pollen allergens with known sequences, 93 (59%) reside in just 5 Pfams,3 suggesting that pollen allergens share a very limited number of relatively unique structures. Identifying the common structural features of allergens may help to elucidate their unique structural elements and potentially the mechanism(s) for their allergenicity. The goal of our research was to use the highly allergenic mountain cedar (Juniperus ashei, Cupressaceae) pollen as a model for characterizing the allergenicity of individual proteins and to identify the structural elements that are required for allergic sensitization or reactions.4 In the current study, we first quantified patient IgE antibodies to crude extract of cedar pollen and to purified Jun a 1,5 a major mountain cedar allergen, using ImmunoCap technology (Phadia, Uppsala, Sweden). The vast majority (median, 93%) of IgE antibodies to cedar pollen in the serum from 35 allergic subjects (34 subjects; see Table E2 in this article’s Online Repository at www.jacionline.org) reacted with Jun a 1 (see Fig E1 in this article’s Online Repository at www.jacionline.org). To define the fine specificity and complexity of these antibodies, we chose 7 sera with high concentrations of IgE anti–Jun a 1 antibodies and adequate serum volume to assess the relative

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FIG 1. Immunoglobulin binding to conformational and linear epitopes of Jun a 1. The % of binding due to discontinuous epitopes is indicated for each serum. These were computed from the relative amount of native (¤) and guanidine-denatured Jun a 1 (B) required to inhibit by 50% the binding of patient IgE to native Jun a 1 on ELISA plates (A) and to induce mast cell degranulation of RBL SX-38 cells sensitized with patient sera (B). RBL, Rat basophilic leukemia.

amount of IgE binding to linear versus discontinuous (conformational) epitopes. We first used ELISA inhibition assays, in which binding of IgE to wells coated with native Jun a 1 was inhibited by prior incubation of the sera with either native or denatured Jun a 1. Our previous study showed that exposing Jun a 1 to 6 mol/L guanidine-HCl caused irreversible denaturation of Jun a 1.6 More than 96% of the IgE reactivity to native Jun a 1 was lost after denaturation of the allergen (Fig 1, A). To assess the biological activity of IgE antibodies to conformational and linear epitopes on Jun a 1,

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we next tested the ability of the same sera to sensitize and induce degranulation of cultured mast cells that express human FcεR1 (RBL SX-38)7 after stimulating with native or denatured Jun a 1. Fig 1, B, shows a dominance of IgE antibodies to conformational epitopes in mast cell degranulation, similar to that seen in ELISAs, indicating that IgE antibodies to conformational epitopes of Jun a 1 should be particularly effective in mediating clinical allergic reactions to cedar pollen. To identify and enumerate the conformational epitopes of Jun a 1 that are recognized by the patients’ IgE, we produced a panel of mAbs and selected those that (1) reacted with native but not denatured Jun a 1 and (2) were inhibited from binding to Jun a 1 by human sera, indicating that these mouse IgG antibodies recognize conformational structures that are also recognized by IgE antibodies in the sera from our highly sensitized patients (see Fig E2 in this article’s Online Repository at www.jacionline. org). Within this panel of antibodies, we identified 4 distinct (noncompeting) groups (G1-G4) on the basis of their cross-inhibition in ELISAs (see Table E1 in this article’s Online Repository at www.jacionline.org). Additional characteristics of these antibodies and the types of epitopes they recognize are shown in Fig E2. Of particular interest was the finding that the epitope for G1 was dependent on a structure maintained by a disulfide bond (Fig E2, B). We have previously reported that guanidine treatment of Jun a 1 results in irreversible loss of a-helical structures.6 Together these observations suggest that epitope G1 and potentially other (G2-G4) conformational epitopes are located in one or both of the a-helical regions, near the N- and C-terminal regions, and contain disulfide bonds (Cys7-Cys21 and Cys285Cys291).8 However, further studies will be required to confirm the importance of these regions and map each of the mAbs to specific conformational elements. To test whether our mAbs G1 to G4 bind to the linear epitopes we previously described, we performed inhibition assays with mAb KW-S91,6 which we have previously mapped to a region of Jun a 1 that contains linear epitopes we termed 2 and 3.9 This antibody caused partial inhibition of E2 and E3 binding (17% and 27% vs 83% for autologous binding of KW-S91). This suggests that the epitope for these mAbs may reside in the extensive parallel helical region of Jun a 1, near the linear epitopes 2 and 3. We next used the mAbs G1 to G4 in competition experiments to determine the frequency with which the IgE antibodies from the 7 subjects recognized G1 to G4 epitopes (Fig 2). Interestingly, the IgE antibody responses of 5 of the 7 sera were strongest against epitopes G2 or G3, suggesting that these may represent dominant epitopes. This pattern is also reflected in the mean intensity of the IgE responses to the 4 epitopes, shown in Fig 2, B. Our findings indicate that the IgE response to mountain cedar pollen is strongly focused on a single protein Jun a 1. The major targets of the patients’ IgE were at least 4 discontinuous regions, brought together by protein folding of this highly dominant allergen. We have also found that purified Jun a 1 is predominantly monomeric (zonal ultracentrifugation indicated >95% monomers; see Fig E3 in this article’s Online Repository at www.jacionline. org). Thus, IgE antibodies from each of our patients’ serum must bind to at least 2 different epitopes10 to degranulate mast cells or basophils. This is consistent with the results of our mAb inhibition studies, which indicate that Jun a 1 displays at least 4 discrete IgE epitopes and that each of our patients’ sera contained antibodies to 2 or more of these discontinuous epitopes.

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FIG 2. Relative binding of patients’ antibodies to discontinuous epitopes defined by mAbs G1 to G4. Binding of serum IgE from 7 patients with cedar pollinosis (a-g) and (A) mean of 7 patients to 4 distinct discontinuous epitopes of Jun a 1 (B). The results indicate the frequency and relative intensity of the patients’ responses to each of the 4 epitopes defined by mAbs G1 to G4.

In conclusion, this study indicates that several conformational epitopes on Jun a 1 are major targets for the IgE response to mountain cedar pollen. This finding suggests that these antibodies are produced in the upper airway after exposure to inhaled, native Jun a 1. This makes Jun a 1 an excellent candidate for the development of new approaches for preventing allergic reactions. These might entail the development of agents that selectively alter the display of dominant epitopes, which may impede epitope spreading. On a more basic level, our findings are consistent with the concept that allergens, as a group, share a relatively small number of structures, which, along with other factors, such as their abundance and stability in the human environment, make them unique among proteins. The characteristics of the allergic response to mountain cedar pollen also make Jun a1 an excellent prototype for identifying the structural basis of allergenicity. We thank Christopher C. Q. Chin, PhD, and J. Ching Lee, PhD, for the ultracentrifugation analysis of Jun a 1. Randall M. Goldblum, MDa Bo Ning, BSa Mark A. Endsley, PhDa D. Mark Estes, DVM, PhDa Barbara M. Judy, PhDa Julius van Bavel, MDb Terumi Midoro-Horiuti, MD, PhDa From the aDepartment of Pediatrics, University of Texas Medical Branch, Galveston, Tex, and bAllergy and Asthma Associates, Austin, Tex. E-mail: [email protected]. This work was supported by the National Institute of Allergy and Infectious Diseases (grant no. R01AI052428 to R.M.G. and grant no. K08AI055792 to T.M.-H.), the American Lung Association (to T.M.-H.), and John Sealy Memorial Endowment Fund from the University of Texas Medical Branch. Disclosure of potential conflict of interest: R. M. Goldblum has received grants from the National Institutes of Health and has received consulting fees, travel support, and payment for lectures from Merck. B. Ning has a patent for antibody-mediated modulation of allergy. T. Midoro-Horiuti has received a grant from the National Institutes of Health (K08AI055792); has received consulting fees, travel support, and payment for lectures from Merck; and has a patent for antibody-mediated modulation of allergy. The rest of the authors declare that they have no relevant conflicts of interest.

REFERENCES 1. Ivanciuc O, Midoro-Horiuti T, Schein CH, Xie L, Hillman GR, Goldblum RM, et al. The property distance index PD predicts peptides that cross-react with IgE antibodies. Mol Immunol 2009;46:873-83. 2. Ivanciuc O, Garcia T, Torres M, Schein CH, Braun W. Characteristic motifs for families of allergenic proteins. Mol Immunol 2009;46:559-68.

3. Radauer C, Breiteneder H. Pollen allergens are restricted to few protein families and show distinct patterns of species distribution. J Allergy Clin Immunol 2006; 117:141-7. 4. Liu Z, Bhattacharyya S, Ning B, Midoro-Horiuti T, Czerwinski EW, Goldblum RM, et al. Plant-expressed recombinant mountain cedar allergen Jun a 1 is allergenic and has limited pectate lyase activity. Int Arch Allergy Immunol 2010; 153:347-58. 5. Midoro-Horiuti T, Goldblum RM, Kurosky A, Goetz DW, Brooks EG. Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:608-12. 6. Varshney S, Goldblum RM, Kearney C, Watanabe M, Midoro-Horiuti T. Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes. Mol Immunol 2007;44:2781-5. 7. Wiegand TW, Williams PB, Dreskin SC, Jouvin MH, Kinet JP, Tasset D. Highaffinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I. J Immunol 1996;157:221-30. 8. Czerwinski EW, Midoro-Horiuti T, White MA, Brooks EG, Goldblum RM. Crystal structure of Jun a 1, the major cedar pollen allergen from Juniperus ashei, reveals a parallel beta-helical core. J Biol Chem 2005;280:3740-6. 9. Midoro-Horiuti T, Mathura V, Schein CH, Braun W, Chin CCQ, Yu S, et al. Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site. Mol Immunol 2003;40:555-62. 10. Scholl I, Kalkura N, Shedziankova Y, Bergmann A, Verdino P, Knittelfelder R, et al. Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice. J Immunol 2005;175:6645-50. Available online June 27, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.009

Systemic and localized seminal plasma hypersensitivity patients exhibit divergent immunologic characteristics To the Editor: Seminal plasma hypersensitivity (SPH) manifests in women as postcoital systemic anaphylaxis and/or localized painful vaginal reactions immediately after intravaginal exposure to seminal fluid.1,2 Systemic SPH has been reported to be an IgE-mediated reaction caused by prostate-specific antigen (PSA), a kallikrein with serine-protease activity. Reactions in women with localized SPH have also been postulated to be IgE mediated because these women are partially or completely responsive to either subcutaneous immunotherapy using fractionated relevant seminal plasma proteins (SPPs) or intravaginal graded challenge using whole seminal plasma (WSP) from their male partners. However, women with localized SPH sometimes differ in their degree of response to treatment.3 The purpose of this investigation was to compare the immunologic responses manifested by women with systemic and localized SPH to further elucidate the

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METHODS Patient selection and serum collection from patients with mountain cedar pollinosis Thirty-five subjects, who had lived for an extended periods of time in the Austin, Texas area, where mountain cedar is a major part of the flora, were recruited on the basis of a diagnosis of mountain cedar allergy (cedar pollinosis) from Dr van Bavel’s clinical and research facilities (Allergy and Asthma Associates, Austin, Tex). Each subject provided serum samples under a UTMB IRB Protocol (06-050) and had a repeat skin prick test with a commercial extract of mountain cedar pollen (Hollister-Stier, Spokane, Wash) if the subject had not been tested in the previous year. Mountain cedar trees pollinate only once a year, usually over a 2- to 3-month period (December to February), and there are very few other plants pollinating at that time. Therefore, the diagnosis of cedar pollinosis was suspected on the basis of clinical symptoms of allergic rhinitis during this season and confirmed by skin prick testing and elevated concentrations of IgE antibodies to mountain cedar pollen by using ImmunoCAP (Phadia).

Purification and chemical modification of Jun a 1 E1

Jun a 1 was purified as we described previously.

Defining the molecular size of native Jun a 1 by ultracentrifugation Velocity sedimentation experiments were performed with an An-60Ti rotor in a Beckman Optima XL-A analytical ultracentrifuge (Palo Alto, Calif) equipped with absorption optics. The molecular weight of Jun a 1 was also estimated by sedimentation equilibrium using the high-speed, meniscus-depletion method.E2 Experiments were conducted at 2 speeds, 15,000 and 19,000 rpm, at 208C. The partial specific volume of Jun a 1 was calculated from the amino acid composition of Jun a 1, using the method of Cohn and Edsall.E3

Quantifying serum IgE antibodies to Jun a 1 and whole cedar pollen extracts We developed an ImmunoCAP assay for quantifying IgE antibodies specifically against Jun a 1. Custom caps were prepared by Phadia, using native Jun a 1 purified in our laboratory. These caps were tested in the Phadia laboratory in Sweden, using known positive and negative sera from our patients and were shown to accurately quantify specific serum IgE antibodies over a wide range of concentrations.

Chemical modification of Jun a 1 For screening mAbs, Jun a 1–coated plates were heated to 378C, 568C, or 758C for 1 hour, or exposed to 6 mol/L guanidine-HCl for 30 minutesE4 to break hydrogen bonds, or reduced with Tris(2-carboxyethyl)phosphine, 5 mM in 50 mM Tris-HCl, 5 mM EDTA, pH 8.2, followed by alkylation with iodoacetamide (5 mM) to reduce and alklyate surface-accessible disulfide bonds. The chemically treated plates were washed with 0.05% Tween-Tris buffered saline (TTBS) to remove any residual chemicals before use in immunoassays. Some of the wells were incubated with anti–Cry j 1 antibody KW-S91, which recognizes a linear sequence shared by Cry j 1 and Jun a 1,E4 followed by HRP-labeled anti-mouse IgG. The amount of KW-91 binding to Jun a 1 after the guanidine treatment of the wells was similar to that for wells coated with native Jun a 1. This indicated that quinidine treatment did not release Jun a 1 from the plate or nonspecifically interrupt the binding of antibodies to the Jun a 1 coat.

ELISAs for comparing the binding of human IgE antibodies to native and denatured Jun a 1 To determine the extent to which individual patient’s IgE react to discontinuous and linear epitopes of Jun a 1, we used ELISA inhibition

assays in which varying amounts of purified native or guanidine-denatured Jun a 1 were added to the sera from 7 patients with high titers of IgE anti–Jun a 1 antibodies (identified in Fig E1) and the residual IgE was assessed by its binding to plates coated with native Jun a 1. Patient sera (1:50 dilution in TTBS) were incubated overnight with various concentrations (5 ng/mL to 50 mg/mL) of either native or guanidine-denatured Jun a 1 or diluent control. The serum-Jun a 1 mixtures were then added to wells coated with native Jun a 1 and incubated for 60 minutes at room temperature. After washing the microtiter plates, the patient IgE bound to the wells was detected using biotinylated-goat antihuman IgE (Vector, Burlingame, Calif), followed by horseradish peroxidase (HRP)-streptavidin conjugates. The results from the colorimetric enzyme assay were plotted against the amount of Jun a 1 (inhibitors) added to the patient sera. Next, the relative amounts of native and denatured Jun a 1 required to reduce the binding of patient IgE to the Jun a 1–coated plate by 50% was computed. This approach allowed us to determine the proportion (%) of IgE antibodies in each serum that recognized discontinuous epitopes.

Mast cell assays comparing the effects of native and denatured Jun a 1 on serum IgE-mediated degranulation A rat basophilic leukemia (RBL), mast cell–like line transfected with a plasmid-expressing human FcεRIa (RBL-SX 38E5) was a kind gift from Dr J-P Kinet. Sera from the 7 patients, selected on the basis of their high titer of IgE anti–Jun a 1 antibodies and availability of adequate volume of their serum for these studies, or control sera without these antibodies were used to sensitize the RBL-SX 38 cell.E6,E7 The RBL-SX38 cells were maintained in Dulbecco modified Eagle medium containing 1.2 mg/mL of G418 (Geneticin, Cellgro, Manassas, Va) until 1 3 105 cells were distributed into each well of 96-well microtiter plates and grown for 48 hours in Dulbecco modified Eagle medium without G148. Sera from the 7 patients, selected on the basis of their high titer of IgE anti–Jun a 1 antibodies, or control sera were added to achieve a final dilution of 1:10 and incubated overnight to allow the serum IgE to bind to the human FcεRIa, thereby sensitizing the mast cells. After washing with Tyrode’s buffer 3 times, 100 mL of Tyrode’s was added to each well and the mast cells were stimulated by adding varying concentrations (0.1-100 ng/mL) of purified native or 6 mol/L guanidine-HCl–denatured Jun a 1. After 30 minutes of incubation at 378C, the release of b-hexosaminidase into the supernatant was quantified, as we have described,E6,E7 and the results were analyzed by determining the concentrations of native and denatured Jun a 1 required to induce equal amounts of b-hexosaminidase release. The relative activity of the native and denatured Jun a 1 was expressed as the % mediator release attributable to conformational epitopes of Jun a 1 (see equation in the Result section for Fig 1, B). Ca21 ionophore (1025 mol/L) and serumsensitized cells cross-linked with antihuman IgE antibodies (Sigma, St Louis, Mo) were used as positive controls and spontaneous release was assessed in wells without cross-linking agents.

Production, screening, selection, and grouping of mAbs to discontinuous epitopes on Jun a 1, which are also recognized by patient sera A panel of mouse mAbs was produced by priming BALB/c mice by subcutaneous injections of Jun a 1 cDNA,E8 prepared as previously described.E9 The cDNA was ligated into the eukaryotic expression vector, pcDNA3.1 (Invitrogen, Grand Island, NY), which had been modified by placing the strong secretion signal sequence from human IL-4 upstream from the ligation site for Jun a 1 cDNA. The purified plasmid (1 mg) in PBS, pH 7.4, was filter sterilized to 0.2 mm and injected into multiple subcutaneous sites 3 times at monthly intervals. The mice then received 2 injections of 10 mg of purified native Jun a 1 protein in incomplete Freund’s adjuvant at monthly intervals. All experiments were approved by our Institutional Animal Care and Use Committee. Spleen cells were harvested and fused with FO cells (SP2/0 myeloma cells, American Type Culture Collection [ATCC]) according to a standard

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protocol.E10 Supernatants from individual wells, containing a single focus of growing hybridoma cells, were tested for antibodies to Jun a 1 by ELISA, using HRP-antimouse IgG, IgA, and IgM as the detector (H&L, 1:1,000, Zymed, Carlsbad, Calif) and orthophenylenediamine (Sigma) and H2O2 as substrates. Hybridoma cells in positive wells were subcloned by limiting dilution at least 3 times. mAbs were then grouped for their similarity in epitope specificity, using ELISA inhibition assays, in which the binding of biotinlabeled IgG from one clone was inhibited by unlabeled culture supernatants from other hybridoma clones.E11 The results of this grouping, as shown in Table E1, indicate that our mAbs to conformational epitopes distributed cleanly into 4 competition groups (G1-G4). Once this grouping was established, we used members within a single group interchangeably. Conformational epitopes on allergens often contain linear ‘‘cores’’ of sequential amino acids. Thus, some of the ‘‘linear’’ human IgE epitopes we described previously using overlapping peptidesE12 might recognize the same sites recognized by 1 or more of the mAbs described here. We tested this hypothesis in cross-inhibition experiments between an mAb (KW-S91) that maps to a region of Jun a 1 containing linear epitopes 2 and 3 and mAbs G1 to G4, which recognize conformational epitopes described in the current Letter to the Editor. The binding of KW-S91 antibody did not inhibit the binding of G1 or G4 antibody to Jun a 1, but weakly inhibited G2 and G3 binding (17% and 27% vs 83% for autologous inhibition by KW-S91 antibody). Our interpretation of these results, as described in the current Letter to the Editor, is that conformational epitopes G2 and G3 may be in proximity with a contiguous region containing linear epitopes 2 and 3. Members of each group (G1-G4) of mAbs were next selected for those that bound to discontinuous epitopes, which were also recognized by serum antibodies from patients with cedar pollinosis. We first screened our antibody groups for reactivity with heat-sensitive epitopes as described above. On the basis of results of these assays, epitopes were divided into heat stable (to 568C and 758C), heat labile (ie, lost activity at 568C and 758C), or partially labile (maintained activity at 568C but not at 758C). Those that were found to be heat labile or partially labile were tested further on Jun a 1–coated wells that had been treated with 6 mol/L guanidine-HCl or reduced and alkylated, as described above.

Competitive ELISAs for determining the frequency with which antibodies in patient serum bind to 4 distinct, discontinuous epitopes of Jun a 1 Jun a 1–coated wells were preincubated with serum from individual patients, diluted 1:4 in TTBS, for 1 hour at room temperature. Next, purified IgG mAb to a discontinuous Jun a 1 epitope was added to patient serum and control wells containing normal human serum and incubated for another 1 hour at room temperature. After washing the wells, the binding of mAb to each of the wells was assessed by the addition of a HRP-conjugated anti-mouse IgG antibody (H&L, Zymed) and results expressed as % inhibition of mAb binding: % inhibition 5 100 3 (1 2 ODwith patient serum/ODwith normal serum).

RESULTS Sedimentation equilibrium results indicate that Jun a 1 is a globular, monomeric glycoprotein Ultracentrifugation was performed to assess the molecular form(s) of purified Jun a 1 molecules in solution. The results of a global fit of all data at both angular velocities yielded molecular weight values of 41,400 6 780 and 42,600 6 800 in 0.53 PBS and 0.05 mol/L Tris-HCl, respectively. Assuming that Jun a 1 has

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similar carbohydrate groups to those described for Cry j 1 from Japanese cedar,E13 the partial specific volume of Jun a 1 would decrease from 0.721 to 0.713. Using a partial specific volume of 0.713, the molecular weight of Jun a 1 in 0.53 PBS and 0.05 mol/L Tris-HCl is 40,200 and 41,400, respectively. This study indicates that Jun a 1 exists as a monomer under these experimental conditions. The sedimentation velocity experiments showed that Jun a 1 acted like a homogeneous species with a sedimentation coefficient, S20,w, of 3.6 and 4.2 in 0.53 PBS and 0.05 mol/L TrisHCl, respectively (Fig E3). The axial ratio of Jun a 1 can then be examined by means of the frictional ratio, f/fo, using Equation 2 in Hesterberg et al.E14 The required information on d, water of hydration, was estimated according to the method of Kuntz,E2 and a value of 0.38 g/g was obtained. A correction factor of 0.7 was applied to d1, as an estimate of the amount of water of hydration of a folded protein.E14 Using known values for the parameters expressed in the equation, f/fo was calculated to be 1.2 and 1.0 in 0.53 PBS and 0.05 mol/L Tris-HCl, respectively. These values indicate that Jun a 1 exists essentially as a globular protein.

REFERENCES E1. Midoro-Horiuti T, Goldblum RM, Kurosky A, Goetz DW, Brooks EG. Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:608-12. E2. Kuntz ID. Hydration of macromolecules, IV: polypeptide conformation in frozen solutions. J Am Chem Soc 1971;93:516-8. E3. Cohn EJ, Edsall JT. Proteins, amino acids and peptides as ions and dipolat ions. New York: Reinhold; 1943:370-81. E4. Varshney S, Goldblum RM, Kearney C, Watanabe M, Midoro-Horiuti T. Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes. Mol Immunol 2007;44:2781-5. E5. Wiegand TW, Williams PB, Dreskin SC, Jouvin MH, Kinet JP, Tasset D. Highaffinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I. J Immunol 1996;157:221-30. E6. Zaitsu M, Narita S, Lambert KC, Grady JJ, Estes DM, Curran EM, et al. Estradiol activates mast cells via a non-genomic estrogen receptor-alpha and calcium influx. Mol Immunol 2007;44:1987-95. E7. Narita S, Goldblum RM, Watson CS, Brooks EG, Estes DM, Curran EM, et al. Environmental estrogens induce mast cell degranulation and enhance IgEmediated release of allergic mediators. Env Health Perspectives 2007;115:48-52. E8. Midoro-Horiuti T, Goldblum RM, Kurosky A, Wood TG, Schein CH, Brooks EG. Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:613-7. E9. Estes DM, Templeton JW, Hunter DM, Adams LG. Production and use of murine monoclonal antibodies reactive with bovine IgM isotype and IgG subisotypes (IgG1, IgG2a and IgG2b) in assessing immunoglobulin levels in serum of cattle. Vet Immunol Immunopathol 1990;25:61-72. E10. Fuller SA, Takahashi M, Hurrell JG. Fusion of myeloma cells with immune spleen cells. Curr Protoc Mol Biol 2001;Chapter 11:Unit11. E11. Bose R, Rector ES, Fischer J, Taronno R, Delespesse G. Production and characterization of mouse monoclonal antibodies to allergenic epitopes on LolpI (Rye I). Immunology 1986;59:309-15. E12. Midoro-Horiuti T, Mathura V, Schein CH, Braun W, Chin CCQ, Yu S, et al. Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site. Mol Immunol 2003;40:555-62. E13. Hino K, Yamamoto S, Sano O, Taniguchi Y, Kohno K, Usui M, et al. Carbohydrate structures of the glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen. J Biochem (Tokyo) 1995;117:289-95. E14. Hesterberg LK, Lee JC, Erickson HP. Structural properties of an active form of rabbit muscle phosphofructokinase. J Biol Chem 1981;256:9724-30.

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FIG E1. IgE binding to pollen extract and purified Jun a 1 IgE. Serum IgE antibodies against whole mountain pollen extracts and purified Jun a 1 for 35 patients with mountain cedar pollinosis were quantified by using ImmunoCap assays (A). The 7 sera selected for further analyses (a-g) are shown by bold line and (-) in Fig E1, A, and individually in B. The relationship of the IgE binding to whole pollen and Jun a 1 is expressed as % Jun a 1 reactivity (Fig E1, B) for each serum.

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FIG E2. mAb binding pattern to native (¤) and denatured (B) Jun a 1. ELISA testing of the relative binding of 4 groups of mAbs (G1-G4) to wells coated with native Jun a 1 (¤) and Jun a 1–coated wells denatured by heating (: 568C, B: 758C, A), wells coated with reduced and alkylated Jun a 1 (B, B), wells coated with Jun a 1 and then treated with 6 mol/L guanidine (B, C). The results indicate that epitopes recognized by mAbs G2 to G4 were disrupted by heat (568C and 758C), while the epitope for mAb G1 was only partially lost after exposure to 758C, but was completely disrupted after reduction and alkylation, which breaks disulfide bonds.

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FIG E3. Molecular mass of Jun a 1. Ultracentrifugation data indicate that Jun a 1 is globular and exists as a monomer with a molecular mass of approximately 42 kDa in a nondenaturing solution. The mass and shape are consistent with glycosylation at several sites.

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TABLE E1. Classification of anti–Jun a 1 mAb by ELISA inhibition Biotinylated mAb Group 1 Unlabeled mAb

Group 1 7g44 3g65 Group 2 39a80 84e78 Group 3 56h58 58g Group 4 70-7 53-44

Group 2

Group 3

Group 4

7g44

3g65

39a80

84e78

56h58

58g

70-7

53-44

83 6 5 81 6 4

80 6 2 79 6 2

363 362

564 364

060 466

365 565

261 060

262 263

262 162

162 363

84 6 5 81 6 6

80 6 7 88 6 6

768 363

14 6 10 564

161 261

462 263

665 060

363 366

262 662

263 564

92 6 6 85 6 12

79 6 3 81 6 4

669 061

763 467

466 664

060 162

162 162

264 362

263 665

564 763

78 6 4 77 6 2

75 6 9 73 6 10

The values are expressed as % inhibition of the binding of biotinylated mAb (columns) to Jun a 1 by the preincubation with excess unlabeled mAb (rows). Values in italics and boldface indicate the autologous inhibition (labeled antibody inhibited by the same antibody without label). The remainder of the numbers indicates that the members of each of these 4 groups do not cross-react with other groups.

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TABLE E2. Characteristics of the study population (n 5 35) No.

Age (y)

Sex

Medication

Cosensitization

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

54 66 46 45 59 29 41 42 35 10 54 25 28 65 38 28 45 54 33 15 46 48

F F M F M F M M F M M F M M F M F F M F F F

Diphenhydramine, loratadine Fexofenadine, fluticasone, olopatadine

Trees, grasses, dust mites Mold, dust mite Oak Dust mite, cat, dog Trees, grasses, ragweeds, cat, dog Grasses, ragweed, mold, dust mite, cat, dog

23 24 25 26 27 28 29 30 31 32 33 34 35

46 32 38 38 39 27 42 21 45 24 47 22 49

M F M F M F F F F F F F F

F, Female; M, male.

Fexofenadine, fluticasone Mometasone, desloratadine Mometasone, desloratadine Mometasone, desloratadine Allergy shots Diphenhydramine, pseudoephedrine Loratadine

Loratadine, fluticasone Fluticasone, diphenhydramine, loratadine Fluticasone

Diseases

Hypertension

Hypertension

Trees, grasses, ragweeds, mold, dust mite Trees, grasses, ragweed, dust mite, mold Grasses, mold, dust mite Pine, grasses, ragweed, mold, dust Elm, ragweed, dust, mold Trees, grasses, ragweed Elm, grasses, ragweed Oak, grasses, ragweed, dust Oak, ragweed, grasses, dust Oak, grasses, ragweed, mold Pecan, pine, ragweed Oak, elm, ragweed, dust mite, mold Oak, grasses, ragweed, mold, dust mite Pecan, oak, ragweed, dust mite Ragweed Oak, elm, grasses, ragweed Grasses, ragweed Ragweed, dust mite

Arthritis, scoriasis, asthma Arthritis, hypertension Asthma

Arthritis, hypertension

Hypertension

Total IgE (kU/L)

110 127 73.5 241 88.4 690 226 151 234 1,209 265 290 50.8 27.8 465 17.7 72.9 65.2 175 137 205 144 73.5 18.7 174 20.6 490 159 691 254 56.4 284 1,020 376 247