Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Histamine-Releasing Factor Is a Novel Alarmin Induced by House Dust Mite Allergen, Cytokines, and Cell Death.

Histamine-releasing factor (HRF) is a multifunctional protein with fundamental intracellular functions controlling cell survival and proliferation. HRF is also secreted during allergic reactions and promotes IgE-mediated activation of mast cells and basophils. In this study, we investigated HRF secretion and its relevance to airway inflammation. HRF monomers were constitutively secreted from BEAS-2B human bronchial epithelial cells (HBECs) and converted to oligomers over the course of culture. Stimulation with house dust mite (HDM) extract increased HRF secretion substantially. Several cytokines involved in asthma pathogenesis showed moderate effects on HRF secretion but dramatically enhanced HDM-induced HRF secretion. HDM-induced HRF secretion from BEAS-2B cells and normal HBECs proceeded via TLR2. Consistent with this, multiple TLR2 ligands, including Der p 2, Der p 5, Der p 13, and Der p 21, induced HRF secretion. Der p 10 (tropomyosin) also promoted HRF secretion. Cell death or incubation with adenosine and ATP, compounds released upon cell death, also enhanced HRF secretion. Furthermore, intranasal administration of recombinant HRF elicited robust airway inflammation in HDM-sensitized mice in an FcεRI-dependent manner. Therefore, we conclude that HRF is a novel alarmin that promotes allergic airway inflammation.

Recombinant T-cell epitope conjugation: A new approach for Dermatophagoides hypoallergen design.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.

Protease allergens as initiators-regulators of allergic inflammation.

Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2-biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE-independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non-protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen-presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease-activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro-Th2 cytokines (IL-6, IL-25, IL-1ß, TSLP) and danger-associated molecular patterns (DAMPs; IL-33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL-33 to produce a super-active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

House dust mite allergy in Malaysia: review of research gaps in the current scenario and the way forward.

The prevalence of house dust mite (HDM) allergy, especially in Asian countries with rapid urbanization, has been increasing. House dust mites thrive in places with relatively high humidity. With the combination of climate change, naturally high humidity, and urbanization, tropical countries like Malaysia are becoming a hotspot for HDM allergy fast. With a previously reported sensitization rate of between 60 and 80%, it is a worrying trend for Malaysia. However, due to incomplete and out-of-date data, as seen by the limited study coverage in the past, these numbers do not paint a complete picture of the true HDM allergy scene in Malaysia. This review briefly discusses the HDM fauna, the HDM sensitization rate, the common diagnosis and therapeutic tools for HDM allergy in Malaysia, and makes suggestions for possible improvements in the future. This review also highlights the need of more comprehensive population-based prevalence studies to be done in Malaysia, encompassing the three main HDMs-Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis-as the lack of up-to-date studies failed to give a clearer picture on the current scenario of HDM allergy in Malaysia. Future studies will be beneficial to the nation in preparing a better blueprint for the management and treatment of HDM allergy.

Zerumbone attenuates house dust mite extract-induced epithelial barrier dysfunction in 16HBE14o- cells.

CONTEXT: The airway epithelial barrier can be disrupted by house dust mite (HDM) allergens leading to allergic airway inflammation. Zerumbone, a natural monocyclic sesquiterpene, was previously found to possess anti-asthmatic effect by modulating Th1/Th2 cytokines. However, the protective role of zerumbone on epithelial barrier function remains to be fully explored. OBJECTIVE: To investigate the effect of zerumbone on HDM extract-induced airway epithelial barrier dysfunction. MATERIALS AND METHODS: Human bronchial epithelial cells 16HBE14o- were incubated with 100 µg/mL HDM extract and treated with non-cytotoxic concentrations of zerumbone (6.25 µM, 12.5 µM, and 25 µM) for 24 h. The epithelial junctional integrity and permeability were evaluated through transepithelial electrical resistance (TEER) and fluorescein isothiocynate (FITC)-Dextran permeability assays, respectively. The localization of junctional proteins, occludin and zona occludens (ZO)-1, was studied using immunofluorescence (IF) while the protein expression was measured by western blot. RESULTS: Zerumbone inhibited changes in junctional integrity (6.25 µM, p ≤ .05; 12.5 µM, p ≤ .001; 25 µM, p ≤ .001) and permeability (6.25 µM, p ≤ .05; 12.5 µM, p ≤ .01; 25 µM, p ≤ .001) triggered by HDM extract in a concentration-dependent manner. This protective effect could be explained by the preservation of occludin (12.5 µM, p ≤ .01 and 25 µM, p ≤ .001) and ZO-1 (12.5 µM, p ≤ .05 and 25 µM, p ≤ .001) localization, rather than the prevention of their cleavage. DISCUSSION AND CONCLUSION: Zerumbone attenuates HDM extract-induced epithelial barrier dysfunction which supports its potential application for the treatment of inflammation-driven airway diseases such as asthma.

Molecular approaches to allergen-specific immunotherapy: Are we so far from clinical implementation?

Conventional allergen-specific immunotherapy (AIT), based on administrations of allergen extracts, represents up to now the unique protocol for the desensitization of allergic patients. Whereas the effectiveness of AIT was evidenced for the treatment of allergic rhinitis and allergic asthma, such strategy remains experimental for food allergies up to now. However, important issues are commonly associated with AIT as the quality of natural allergen extracts, the long duration and adverse side-effects which negatively affect successful desensitization together with the patient compliance. The rapid progression of molecular allergology made possible the quest of safer, shorter and more effective immunotherapeutic approaches. The aim of this review was to provide an update on these different innovative recombinant derivatives including their efficacy but also their limitations. Despite promising preclinical and early clinical studies, the absence of convincing data in large phase III trials precludes so far the translation of these immunotherapeutic candidates into the clinic.

Proteolytic, lipidergic and polysaccharide molecular recognition shape innate responses to house dust mite allergens.

House dust mites (HDMs) are sources of an extensive repertoire of allergens responsible for a range of allergic conditions. Technological advances have accelerated the identification of these allergens and characterized their putative roles within HDMs. Understanding their functional bioactivities is illuminating how they interact with the immune system to cause disease and how interrelations between them are essential to maximize allergic responses. Two types of allergen bioactivity, namely proteolysis and peptidolipid/lipid binding, elicit IgE and stimulate bystander responses to unrelated allergens. Much of this influence arises from Toll-like receptor (TLR) 4 or TLR2 signalling and, in the case of protease allergens, the activation of additional pleiotropic effectors with strong disease linkage. Of related interest is the interaction of HDM allergens with common components of the house dust matrix, through either their binding to allergens or their autonomous modulation of immune receptors. Herein, we provide a contemporary view of how proteolysis, lipid-binding activity and interactions with polysaccharides and polysaccharide molecular recognition systems coordinate the principal responses which underlie allergy. The power of the catalytically competent group 1 HDM protease allergen component is demonstrated by a review of disclosures surrounding the efficacy of novel inhibitors produced by structure-based design.

The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.

Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates.

House dust mite (HDM) protease allergens, through cleavages of critical surface proteins, drastically influence the initiation of the Th2 type immune responses. However, few human protein substrates for HDM proteases have been identified so far, mainly by applying time-consuming target-specific individual studies. Therefore, the identification of substrate repertoires for HDM proteases would represent an unprecedented key step toward a better understanding of the mechanism of HDM allergic response. In this study, phage display screenings using totally or partially randomized nonameric peptide substrate libraries were performed to characterize the extended substrate specificities (P5-P4') of the HDM proteases Der p 1, Der p 3 and Der p 6. The bioinformatics interface PoPS (Prediction of Protease Specificity) was then applied to define the proteolytic specificity profile of each protease and to predict new protein substrates within the human cell surface proteome, with a special focus on immune receptors. Specificity profiling showed that the nature of residues in P1 but also downstream the cleavage sites (P' positions) are important for effective cleavages by all three HDM proteases. Strikingly, Der p 1 and Der p 3 display partially overlapping specificities. Analysis with PoPS interface predicted 50 new targets for the HDM proteases, including 21 cell surface receptors whose extracellular domains are potentially cleaved by Der p 1, Der p 3 and/or Der p 6. Twelve protein substrate candidates were confirmed by phage ELISA (enzyme linked immunosorbent assay). This extensive study of the natural protein substrate specificities of the HDM protease allergens unveils new cell surface target receptors for a better understanding on the role of these proteases in the HDM allergic response and paves the way for the design of specific protease inhibitors for future anti-allergic treatments.

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.