The nanotopography of SiO2 particles impacts the selectivity and 3D fold of bound allergens.

A detailed description of the changes that occur during the formation of protein corona represents a fundamental question in nanoscience, given that it not only impacts the behaviour of nanoparticles but also affects the bound proteins. Relevant questions include whether proteins selectively bind particles, whether a specific orientation is preferred for binding, and whether particle binding leads to a modulation of their 3D fold. For allergens, it is important to answer these questions given that all these effects can modify the allergenic response of atopic individuals. These potential impacts on the bound allergen are closely related to the specific properties of the involved nanoparticles. One important property influencing the formation of protein corona is the nanotopography of the particles. Herein, we studied the effect of nanoparticle porosity on allergen binding using mesoporous and non-porous SiO2 NPs. We investigated (i) the selectivity of allergen binding from a mixture such as crude pollen extract, (ii) whether allergen binding results in a preferred orientation, (iii) the influence of binding on the conformation of the allergen, and (iv) how the binding affects the allergenic response. Nanotopography was found to play a major role in the formation of protein corona, impacting the physicochemical and biological properties of the NP-bound allergen. The porosity of the surface of the SiO2 nanoparticles resulted in a higher binding capacity with pronounced selectivity for (preferentially) binding the major birch pollen allergen Bet v 1. Furthermore, the binding of Bet v 1 to the mesoporous rather than the non-porous SiO2 nanoparticles influenced the 3D fold of the protein, resulting in at least partial unfolding. Consequently, this conformational change influenced the allergenic response, as observed by mediator release assays employing the sera of patients and immune effector cells. For an in-depth understanding of the bio-nano interactions, the properties of the particles need to be considered not only regarding the identity and morphology of the material, but also their nanotopography, given that porosity may greatly influence the structure, and hence the biological behaviour of the bound proteins. Thus, thorough structural investigations upon the formation of protein corona are important when considering immunological outcomes, as particle binding can influence the allergenic response elicited by the bound allergen.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

TGFß1 mimetic peptide modulates immune response to grass pollen allergens in mice.

BACKGROUND: Transforming growth factor ß1 (TGFß1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFß1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFß1 mimetic peptide (TGFß1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. METHODS: The in vitro action of TGFß1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice. RESULTS: In vitro, TGFß1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFß1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFß1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. CONCLUSION: The TGFß1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.

Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.