Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG1/IgG2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT.

Birch pollen (BP) is a clinically relevant aeroallergen source affecting up to 16% of the European population. In predisposed individuals, BP exposure triggers Th2 immune responses that orchestrate allergic sensitization.1 The key cytokine for Th2 differentiation, interleukin-4 (IL-4), binds with high affinity to IL-4R alpha (IL-4Rα) to form the active type-I IL-4 receptor with the common gamma (γC) chain. Downstream, induced GATA3 mainly regulates the expression of type 2 cytokines. Although the initiation of Th2 differentiation by pollen sources is still unclear, the reported adjuvant function of specific BP-derived compounds other than the major allergen2, 3 suggests that Th2-inducing signals originate from the BP source itself. We, therefore, aimed to study the role of the IL-4/IL-4R pathway in BP-induced Th2 responses by (i) investigating the ability of BP extracts (BPE) compared with other pollen species to bind IL-4Rɑ via ELISA, (ii) screening for latent IL-4Rɑ ligands by fractionating BPE via size exclusion chromatography (SEC), and (iii) assessing associated Th2 responses in vivo using IL-4 reporter mice. Finally, (iv) the functional downstream signaling elicited by the interaction with the IL-4R was examined in pilot in vitro assays. Commercial (c)BPE showed a dose-dependent binding activity to IL-4Rα (Figure 1A), which was not attributable to the major allergen, Bet v 1 (Figure 1B). The activity of the natural ligand IL-4 was higher than for cBPE. By contrast, neither timothy grass (cGPE) nor short ragweed (cRPE) pollen extracts exhibited this IL-4Rα-binding activity (Figure 1A; Figure S1-S3A), indicating a BP-specific effect. Proteinase K-mediated degradation of cBPE (PK-BPE) abolished the binding signal (Figure 1C; Figure S1-S3B), inferring a protein ligand. In inhibition and competition approaches, cBPE was able to block the binding of IL-4Rα to IL-4 dose-dependently and, conversely, IL-4 hindered IL-4Rα to interact with cBPE (Figure 1D,E; Figure S1-S3C), implying similar binding interaction. Interestingly, BPE from self-collected pollen (sBPE) displayed a stronger binding activity than cBPE (Figure 2A). Via interpolation from an IL-4 standard curve, an IL-4Rα-binding activity of 37% for sBPE versus 20% for cBPE was measured. SEC-generated fractions of sBPE (sF1-7) and cBPE (cF1-5) both showed an increased binding activity from low (LMW) to high (HMW) molecular weight fractions. The augmented binding activity observed for sF4-sF1 (86, 65, 55, 39%) and cF1 (47%) suggests the presence of a HMW ligand (Figure 2A; Figure S1-S3). Consistently, compared with original cBPE, only the HMW fraction significantly induced a Th2 response in vivo, measured by the induction of IL-4/eGFP expression in CD4-positive (+) lymphocytes (Figure 2B). Immunization with sBPE similarly induced a significant increase in 13% IL-4/eGFP+CD4+ cells (Figure 2C). Blocking of IL-4Rα on sBPE-stimulated human naïve CD4+T cells increased anti-inflammatory IL-10 secretion, and in HEK reporter cells cBPE stimulation interfered with IL-4-induced STAT6 phosphorylation (Figure S1-S3A,B), preliminarily suggesting the activation of a non-canonical pathway downstream of IL-4R. BPE was demonstrated earlier to act on T cells favoring IL-5 and IL-13 expression compared with Th1-associated TNFα and IL-2.3 IL-4R signaling was also shown to regulate IL-10 secretion to establish Th2 dominance4 and IL-4Rα-deficiency in FoxP3+Tregs exacerbated airway inflammation in a house dust mite sensitization model.5 By exploring the existence of intrinsic Th2 inducers within BPE, our results revealed the peculiar feature of BP-derived compound(s) contained in the HMW fraction and affected by protein degradation to interact with IL-4Rα, associated with the induction of Th2 response in vivo. Several microbial as well as plant-derived cytokine mimics with immunomodulating functions have already been described,6 yet this is the first study to present a natural IL-4Rα ligand other than IL-4. Considering IL-4R expression on various immune cells, concrete downstream effects mediated by BP via the IL-4R pathway to prime Th2 response in vivo remains to be investigated. The identification of BP-intrinsic immunostimulators is relevant to better understand the mechanisms initiating BP allergy and will provide novel therapeutic targets. The authors thank Univ. Prof. Dr. Fatima Ferreira of the Department of Biosciences and Medical Biology for her great support endorsed by productive discussions and providing lab equipment. The research was supported by Austrian/German Joint Science Funds (FWF/DFG Project I5312), by the University of Salzburg priority program Allergy-Cancer-BioNano Research Centre, by the doctoral program Immunity in Cancer and Allergy—ICA funded by the Austrian Science Fund (FWF W1213) and by the Doctoral School Program Biomolecules of the University of Salzburg. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Appendix S1. Figures S1-S3. 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.

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of β-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

The mechanisms involved in the induction of allergic sensitization by pollen are not fully understood. Within the last few decades, findings from epidemiological and experimental studies support the notion that allergic sensitization is not only dependent on the genetics of the host and environmental factors, but also on intrinsic features of the allergenic source itself. In this review, we summarize the current concepts and newest advances in research focusing on the initial mechanisms inducing pollen sensitization. Pollen allergens are embedded in a complex and heterogeneous matrix composed of a myriad of bioactive molecules that are co-delivered during the allergic sensitization. Surprisingly, several purified allergens were shown to lack inherent sensitizing potential. Thus, growing evidence supports an essential role of pollen-derived components co-delivered with the allergens in the initiation of allergic sensitization. The pollen matrix, which is composed by intrinsic molecules (e.g. proteins, metabolites, lipids, carbohydrates) and extrinsic compounds (e.g. viruses, particles from air pollutants, pollen-linked microbiome), provide a specific context for the allergen and has been proposed as a determinant of Th2 polarization. In addition, the involvement of various pattern recognition receptors (PRRs), secreted alarmins, innate immune cells, and the dependency of DCs in driving pollen-induced Th2 inflammatory processes suggest that allergic sensitization to pollen most likely results from particular combinations of pollen-specific signals rather than from a common determinant of allergenicity. The exact identification and characterization of such pollen-derived Th2-polarizing molecules should provide mechanistic insights into Th2 polarization and pave the way for novel preventive and therapeutic strategies against pollen allergies.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients’ first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.