Amb a 1 isoforms: Unequal siblings with distinct immunological features

Abstract Background Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunological characteristics of Amb a 1 isoforms are not fully investigated. Here, we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms. Methods After purification, the isoforms were physicochemically characterized, tested for antibody binding and induction of human T‐cell proliferative responses. Their immunological properties were further evaluated in vitro and in vivo in a mouse model. Results Amb a 1 isoforms exhibited distinct patterns of IgE binding and immunogenicity. Compared to Amb a 1.02 or 03 isoforms, Amb a 1.01 showed higher IgE‐binding activity. Isoforms 01 and 03 were the most potent stimulators of patients’ T cells. In a mouse model of immunization, Amb a 1.01 induced higher levels of IgG and IgE antibodies when compared to isoforms 02 and 03. Interestingly, ragweed‐sensitized patients also displayed an IgG response to Amb a 1 isoforms. However, unlike therapy‐induced antibodies, sensitization‐induced IgG did not show IgE‐blocking activity. Conclusion The present study showed that naturally occurring isoforms of Amb a 1 possess different immunogenic and sensitizing properties. These findings should be considered when selecting sequences for molecule‐based diagnosis and therapy for ragweed allergy. Due to its high IgE‐binding activity, isoform Amb a 1.01 should be included in diagnostic tests. In contrast, due to their limited B‐ and T‐cell cross‐reactivity patterns, a combination of different isoforms might be a more attractive strategy for ragweed immunotherapy.


| INTRODUCTION
Worldwide, pollen of short ragweed is recognized as a major allergy risk for atopic individuals. Epidemiological studies showed that 23% to 32.8% of the US population is sensitized to ragweed, whereas prevalence of sensitization in European countries varies between 3.5% (eg, Italy) and 54% (eg, Hungary). [1][2][3] A study in northern China showed that 6.5% of allergic patients responded positive to ragweed pollen in skin prick tests. 4 In South America, 23% of Colombian patients with acute asthma displayed ragweed-specific IgE antibodies. 5 More than 95% of ragweed pollen-allergic patients display IgE antibodies against the major allergen Amb a 1, which is a member of the pectate lyase family. 6 Allergenic pectate lyases have also been identified as major allergens in the pollen of Cupressaceae trees such as Mediterranean cypress, mountain cedar, as well as Japanese cedar and cypress. Interestingly, IgE cross-reactivity between ragweed and Cupressaceae pollen-derived pectate lyases has been reported to be relatively low. 7 To date, five different Amb a 1 isoforms have been acknowledged by the WHO/IUIS allergen nomenclature subcommittee (www.allergen.org) showing sequence homologies between 63% and 87% (Fig. S1B). 8 Previous studies showed that isoforms of certain major allergens display distinct immunological properties. For example, isoforms of the major house dust mite allergen Der p 2 diverge only by 3% in their amino acid sequences, but were shown to differ in their IgEbinding properties and to induce different cytokine patterns upon stimulation of PBMCs from allergic and nonallergic donors. 9 Similarly, two isoforms of Bet v 1 with sequence identity of 96% showed striking differences in their IgE-binding properties and in their capacity to activate T cells from allergic patients. 10 These differences seem to be linked to the capacity of Bet v 1 isoform 0102 to form cysteine-linked aggregates 11 and its fold dynamics. These properties, which were demonstrated to be critical for binding to cathepsin S and for efficient processing, 12 are not shared by the Bet v 1.0101 isoform.
Recent reports suggested that Amb a 1 isoforms might display distinct antibody binding properties. 6,8 This prompted us to analyze in great detail the allergenic as well as immunogenic properties of Amb a 1 isoforms. Understanding the immunological and allergenic properties of individual isoforms comprising natural Amb a 1 is of major importance for the development of adequate and efficient products for diagnosis and therapy of ragweed pollen allergy.

| Protein purification
Natural Amb a 1 was purified from 6 g of Ambrosia artemisiifolia pollen (Batch: 020511204 purchased from Allergon AB, € Angelholm, Sweden). Recombinant Amb a 1.03 was produced in the yeast Pichia pastoris and purified from culture supernatants. Methods are described in detail in Appendix S1.

| Peptide analysis by nano-LC-MS/MS
Analyses of tryptic peptides obtained from ragweed pollen extracts and from purified isoforms were performed as described in Appendix S1.

| Physicochemical characterization
Physicochemical analyses were performed as described in Appendix S1.

| Patients 0 sera
Ragweed-allergic patients included in the study were identified based on typical case history, positive skin prick test, and ImmunoCAP analysis (CAP w1 common ragweed Ambrosia artemisiifolia, Phadia, AB, Uppsala, Sweden) (Table S1). Experiments using anonymized serum samples from allergic patients were approved by the ethics committee of the Medical University of Vienna (Nr. 712/2010).

| Proliferation assays: PBMC and TCC
T-cell recognition of Amb a 1 isoforms was assessed using peripheral blood mononuclear cells (PBMCs) and Amb a 1-specific T-cell clones (TCC) isolated from ragweed-allergic patients. 13 A detailed description is provided in Appendix S1. Experiments using anonymized blood samples from allergic patients were approved by the ethics committee of the Medical University of Vienna (Nr. 497/2005).

| Immunoblots and ELISA
Immunoblots and ELISA experiments were performed according to the established protocols. A detailed description is provided in Appendix S1.

| Mediator release assays
Mediator release assays were performed using RBL-2H3 cells transfected with the alpha chain of the human IgE receptor 14 and passively sensitized with IgE antibodies from ragweed-allergic donors as described in Appendix S1.

| Facilitated antigen-binding assay (FAB)
To determine Amb a 1 isoform-specific blocking capacity of patients' IgG and IgG of immunized mice toward Amb a 1-specific human IgE, the FAB assay 15,16 was used. A detailed description is given in Appendix S1.

| Animal experiments
Female BALB/c mice (Janvier Labs, Saint-Berthevin, France) at the age of 8 weeks were used for experiments (five animals/group). 5 lg of antigen was either adsorbed to 50% Alu-Gel-S (Serva, Heidelberg,

| Purification and physicochemical characterization of Amb a 1 isoforms
Isoforms 01, 02, and 03 were purified from ragweed pollen extracts by standard chromatography. Isoform 03 was also produced as a recombinant protein in P. pastoris. The purified Amb a 1 isoforms were able to inhibit between 59% and 87% of the IgE binding to ragweed pollen extracts, as determined in inhibition ELISA and immunoblot experiments ( Figure 1B,C). After purification, the identity, purity, and isoform composition of the preparations were confirmed by mass spectrometry (Fig. S2). The nAmb a 1.01 preparation was found to consist of 94% isoform 01, 4% isoform 04, and 1% isoform 02. The nAmb a 1.02 preparation consisted of 86% isoform 02 and 12% isoform 01. The natural preparation of Amb a 1.03 contained 87% of isoform 03 and 8% of isoform 01. Recombinant isoform 03 had a purity of >99%.
Using sera from ragweed-allergic patients in immunoblots, crossinhibition ELISA, and mediator release assays, no significant differences in the IgE-binding properties of recombinant and natural isoform 03 could be detected (Fig. S3). Due to low purification yields of the natural form (40 lg/g pollen), the recombinant form of isoform 03 was selected for further experiments. As determined by the HEK-Blue TM mTLR4 reporter cell assay, the levels of endotoxin in isoforms 01 and 03 preparations were below the detection limit of 6.1 EU/mg, whereas for Amb a 1.02 a value of 166 EU/mg was measured.
The secondary structure elements of the three Amb a 1 isoforms were analyzed using CD and FTIR (Fig. S4). The CD spectra showed the typical shape of proteins with high beta-sheet content in combi-  F I G U R E 1 IgE binding to Amb a 1 isoforms analyzed by cross-inhibition ELISA to coated Amb a 1 isoforms (A) and ragweed extract (B). IgE immunoblot of ragweed extract using a serum pool from 10 allergic patients with (+) or without (À) preincubation with a mixture of equal amounts of Amb a 1 isoforms 01, 02, and 03 (C). The arrows indicate full-length natural Amb a 1 and the high IgE-binding beta chain, respectively. The beta chain is produced by proteolysis of Amb a 1 during pollen extraction detected (Figure 2A-C). However, in mediator release assays, isoform 01 was found to react significantly stronger than the other isoforms.

| Amb a 1-specific IgG4 antibodies from non-AIT patients do not show IgE-blocking activity
The IgE-blocking activity of sensitization-versus therapy-induced IgG antibodies was evaluated using the FAB assay. We selected five non-AIT patients and compared them with seven AIT patients. All patients (non-AIT and AIT) showed high levels of Amb a 1-specific IgG4 recognizing each isoform. In six of seven AIT patients, IgGblocking activity could be detected, but their blocking capacity did not correlate with IgG antibody levels. Remarkably, in contrast to therapy-induced IgGs, sensitization-induced specific IgGs showed no IgE-blocking activity ( Figure 2E,F).

| Amb a 1.01 shows high immunogenicity in mice
To investigate the immunological properties of Amb a 1 isoforms in vivo, mice were immunized using two immunization schedules, with and without ALUM as adjuvant ( Figure 3A). In both immunization protocols, isoform 01 was the most potent antigen inducing the highest IgG1 titers. Moreover, the onset of the immune response against isoform 01 was faster compared to the other tested antigens.
In the presence of ALUM, immunization with isoform 03 induced higher IgG2a titers than with isoforms 01 or 02, whereas in the absence of ALUM, higher titers were observed with isoform 01 (Fig-ure 3B,C; Table S3).

| Amb a 1.01 isoform is a very potent allergen
In mediator release assays, we found that adsorption to ALUM had only minor effects on the induction of IgE specific for iso-

| Proliferation assays with PBMC
Amb a 1 isoforms were tested for their capacity to induce proliferation in PBMCs from seven ragweed-allergic patients. PBMCs were stimulated with three different concentrations (0.6, 1.25, and 2.5 lg/ mL) of Amb a 1 isoforms. Higher and stronger proliferative responses were obtained upon stimulation with Amb a 1.01 and 1.03, when compared with Amb a 1.02 ( Figure 5B).

| Amb a 1.03-specific T-cell clones
Five Amb a 1.03-reactive TCC obtained from three different ragweed-allergic donors were specific for five independent epitope regions of Amb a 1.03 (epitope sequences are indicated in Figure 5A). 13 Four of five TCC also reacted to Amb a 1.02 and only two of five to Amb a 1.01. These results suggest that at the T-cell level, isoforms 02 and 03 have the highest cross-reactivity level, in conformity with their higher sequence identity.

| Mouse antibodies block human IgE binding to Amb a 1 in an isoform-specific manner
To analyze the epitope profile and IgE-blocking capacity of mouse antibodies induced by immunization with Amb a 1 isoforms, we performed FAB assays (schematic in Figure 6A). Mouse sera were d14 d28 d14 d28 d14 d28 Similarly, sera from isoforms 02-and 03-immunized animals achieved almost 100% blocking activity against their respective complexes (96.3% and 95.6%, respectively). In contrast, sera from isoforms 02and 03-immunized mice showed very limited capacity (28.6% and 7.7%, respectively) to block the formation of IgE-isoform 01 complexes. Sera from isoform 01-immunized mice showed very low blocking activity against isoforms 02 and 03 (24.8% and 3.6%, respectively).
Experiments using combinations of isoform-specific mouse sera showed that only an equal mix of sera containing antibodies against all three isoforms resulted in high blocking capacity ( Figure 6B).

| DISCUSSION
Amb a 1, the most abundant allergen in ragweed pollen, is composed of a mixture of five isoforms with amino acid sequence identities ranging between 63% and 87%. The clinical relevance of Amb a 1 has been documented in numerous publications: (i) A study by Zeiss et al. 18 showed that as much as 13% of the total serum IgE in ragweed-allergic patients is specific for Amb a 1; (ii) the content of Amb a 1 (formerly antigen E) in pollen extracts nicely correlates with the potency of the extract, as determined by skin prick tests in ragweed-allergic patients 19 ; (iii) immunotherapy with purified Amb a 1 was shown to be as effective as whole ragweed extract in reducing symptoms induced by pollen exposure. 20   allergens Amb a 8 and Amb a 9 display very low IgE inhibition capacity toward coated ragweed pollen extract. In these experiments, isoforms 01 and 02 showed the highest capacity to inhibit IgE binding (up to 87% of total ragweed-specific IgE) to coated ragweed pollen extract. As our results presented here as well as published data showed that Amb a 1 isoforms 01, 02, and 03 bind most of the patients 0 ragweed-specific IgE, 8,21 our study focused on the characterization of these three isoforms.
Amb a 1-allergic patients receiving AIT showed high levels of IgG (especially IgG4) against all Amb a 1 isoforms, which is an expected effect of the therapy. However, many of the non-AIT patients showed Amb a 1-specific IgG1 and more surprisingly also IgG4 antibodies. Recent studies with large birth cohorts have also found IgG antibodies to a wide variety of allergens. 22,23 However, in the study by Schwarz et al., IgG4 antibodies against airborne allergens were rarely found. 22 In this respect, it should be noted that in the MAS birth cohort study, antibodies have been tested at the age of 2 years. 22 This could provide an explanation for discrepancies on the detection of allergen-specific IgG4 antibodies, as IgG4 antibody production seems to depend on repeated antigen stimulation for longer periods of time. 24 Nevertheless, the occurrence of allergen-specific IgG4 in treated and nontreated ragweed-allergic patients raises the question on the beneficial effects (eg, blocking activity) attributed to this antibody class in immunotherapy. 25 Our results using functional      F I G U R E 6 Schematic representation of the FAB assay (A) The inhibitory capacity of mouse immunization-induced IgG antibodies was tested with sera of Amb a 1.01-, Amb a 1.02-, or Amb a 1.03-immunized mice with the respective Amb a 1 isoforms. A pool of ragweedallergic patients 0 sera was used as an indicator for the IgE complex formation (B)