Bet v 1 from birch pollen is a hypoallergen with vitamin D3 in the pocket
Authors: Karin Hufnagl, Livia Kromp, Rodolfo Bianchini, Sheriene Moussa Afify, Markus Wiederstein, Frank A. Redegeld, Iveta Zuvalova, Zdenek Dvorak, Gerlinde Hofstetter, Franziska Roth‐Walter, Luis F. Pacios, Erika Jensen‐Jarolim
Journal: Allergy (2021)

Based on our previous molecular studies with ligands for lipocalin and lipocalin-like proteins, we predicted that the active vitamin D metabolite vitamin D3 (VD3) could bind into the intramolecular pocket of the major birch pollen allergen Bet v 1 (Bet v 1.0101),1, 2 transforming it from apo (empty) to holo-Bet v 1. As VD3 per se is able to modulate innate and adaptive immune responses,3 we hypothesized that the binding of this immunomodulatory molecule would modify the allergenicity of Bet v 1. In a first step, we analysed VD3 binding to Bet v 1 by in silico docking analysis, in vitro ANS competition assay and IZ-VDRE cell reporter assay (Figure 1). In silico calculations based on the crystal structure of the Bet v 1-naringenin complex (PBD entry 4A87) (Figure 1A) rendered an affinity energy of −9.9 kcal/mol, corresponding to a dissociation constant of 0.055 µmol/L. The calculated affinity of VD3 to Bet v 1, being thus 6-times stronger than of vitamin A metabolite retinoic acid,1 could be supported by in vitro assays. First, in an ANS competition assay, VD3 dose-dependently displaced ANS from Bet v 1 (Figure 1B). For the second assay, we used the human IZ-VDRE cell line stably transfected with vitamin D response elements VDRE-I from the human CYP24A1 promoter and expressing the gene for luciferase under the control of VDRE.4 The luciferase activity was dose-dependently activated by VD3 (Figure S1), ensuring the functionality of the assay. Incubation of IZ-VDRE cells with holo-Bet v 1 (preincubated with 1 µmol or 0.1 µmol VD3) induced luciferase expression comparable to the signal from the positive control VD3 alone, while empty apo-Bet v 1 did not elicit luciferase activity (Figure 1B). We hypothesized that shuttling of VD3 via Bet v 1 induced the luciferase activity, although we cannot exclude intracellular dissociation of VD3 from Bet v 1. Nevertheless, our results so far proposed that Bet v 1 is able to bind VD3 into its intramolecular cavity in vitro. We then investigated the influence of VD3 in holo-Bet v 1 on the cellular immune response. Stimulation of PBMCs from birch pollen (BP)-allergic donors with apo-Bet v 1 induced significant upregulation of Th1 (IFNγ) and Th2 (IL-10, IL-13) cytokines in supernatants compared to medium control (Figure 2A). In contrast, holo-Bet v 1-treated cells displayed significantly decreased IFNγ, IL-10 and IL-13 levels in comparison with apo-Bet v 1 stimulated cells, similar to treatment with VD3 alone (Figure 2A). When transported in holo-Bet v 1, VD3 more than VD3 alone, reduced the expression of co-stimulatory molecules HLA-DR+ and CD86+ on CD14+ monocytes, indicating their reduced activation status (Figure 2B left). Remarkably, in the T-cell compartment, only holo-Bet v 1 treatment led to significant reduction of CD3+CD4+ cells expressing the Th2 marker CRTh2 (Figure 2B right). Prostaglandin D2 receptor CRTh2 is expressed by Th2 lymphocytes, eosinophils, basophils and innate lymphoid cells (ILC2s) and involved in recruitment of these cell types and Th2 cytokine production during allergic response.5 The CD3+CD4+CD25+CD127- regulatory T cells were significantly increased by both apo- and holo-Bet v 1 treatments (Figure 2B middle). In the B-cell compartment, we found significantly reduced numbers of CD19+ B cells after treatment with the holo- but not with the apo-form of Bet v 1 (Figure 2C left). Reduction of IgE committed B cells may be one mechanism why VD3 previously inhibited IgE synthesis in vitro.6 The holo-Bet v 1 also showed significantly lower IgE-binding capacity, at least in some patients, compared to the apo-allergen (Figure 2C middle), and it induced significantly less mediator release from primary human mast cells pre-sensitized with serum IgE of BP-allergic donors (Figure 2C right; Figures S2 and S3). Based on our in silico structural analyses, the IgE-specific effects may be due to epitope masking by the VD3 ligand of two previously described IgE-binding B-cell epitope regions in the molecular surface of Bet v 1 (Figure 1C). Thus, our data for the first time revealed VD3 as a novel ligand in the pocket of the major birch pollen allergen Bet v 1, and that VD3 is able to down-regulate the allergic immune response to Bet v 1. We evidence that holo-Bet v 1 shuttles VD3 into immune cells, prevents Th2 responses to Bet v 1 in vitro by reducing CRTh2+ Th2- and CD19+ B cells, inhibits antigen presentation and reduces production of inflammatory cytokines. The present data are in line with our findings that loading of Bet v 1 allergen with vitamin A metabolite induces immune tolerance.1 The principle of vitamin spiking of allergens could be of use to improve immunotherapeutic approaches in future, and at much lower doses than recently suggested.7 We thank Bart R. Blokhuis for excellent technical assistance. EJJ, LFP and FRW are inventors of EP2894478, owned by Biomedical International R+D GmbH, Vienna, Austria, of which EJJ is shareholder. SMA was supported by a grant of Biomedical Int. R+D GmbH and by Bencard Allergy GmbH. The other authors declare they have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.