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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.

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins’ structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO2 nanoparticles with Alhydrogel® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens’ structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.

Advancements in hybridoma technology have enabled the production of human IgE monoclonal antibodies (hIgE mAb) for successful IgE epitope mapping of major allergens. Here, we assessed the hypoallergenicity of three IgE-epitope mutants (single 4C8 or 2F10, and double 4C8 + 2F10 epitope mutants) of house dust mite allergen (HDM) Der p 2. Humanized rat basophilic leukemia (huRBL) cells, passively sensitized overnight with either pairs of Der p 2 specific hIgE mAb (2F10, 4C8 or 2G1) or HDM-allergic serum (n=8), were stimulated with either wildtype (WT) Der p 2 or an epitope mutant and mediator release was measured. No degranulation was induced upon stimulation with all mutants, when cells were sensitized with pairs of hIgE mAb specific for at least one mutated epitope. HIgE mAb specific for non-mutated epitopes led to mediator release comparable to WT Der p 2, indicating that epitopes recognized by the three different hIgE mAb are not overlapping and that the 3D-structure of the mutants is conserved. The double 4C8 + 2F10 epitope mutant had a significantly reduced maximal mediator release (48.3%) compared to the WT, in cells sensitized with allergic donor serum. Overall, the area-under-the-curve of mediator release curves induced by the mutants was significantly lower (31-65%) compared to WT. When comparing the EC , the double 4C8 + 2F10 epitope mutant required a 158-fold higher antigen concentration to induce the same extent of mediator release as WT Der p 2. Der p 2 epitope mutants display significantly reduced allergenicity. Particularly, the double 4C8 + 2F10 epitope mutant demonstrated a strong potential as a novel AIT vaccine candidate.

Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release. One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

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.

Immunity to Ascaris lumbricoides influences the pathogenesis of allergic diseases. Antibody responses to its proteins have been found to be associated with asthma presentation; however, helminth products that induce immunosuppression have been reported, which also raise specific antibodies. We aimed to evaluate antibody responses (IgE, IgG4 and IgG) to two A. lumbricoides molecules, Asc l 5 and Al-CPI (an anti-inflammatory Cysteine Protease Inhibitor), in an endemic population, exploring their relationships with the infection and asthma. The two molecules were produced as recombinant proteins in E. coli expression systems. Specific antibodies were detected by ELISA. Lower human IgE, but higher IgG4 and IgG antibody levels were observed for Al-CPI than for rAsc l 5. The IgE/IgG4 isotype ratio was significantly higher for Asc l 5 than for Al-CPI. In humans Al-CPI did not induce basophil activation as has been previously described for Asc l 5. In mice, Al-CPI induced fewer IgE responses, but more IgG2a antibody titers than rAsc l 5. Our results suggest that these molecules elicit different patterns of immune response to A. lumbricoides.

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 (10 to 10 ) 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.

[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].

Birch pollen allergy affects more than 20% of the European allergic population. On a molecular level, birch pollen allergy can be linked to the two dominant allergens Bet v 1 and Bet v 2. Bet v 2 belongs to the profilin family, which is abundant in the plant kingdom. Importantly, the homologous plant profilins have a conserved cysteine motif with a currently unknown functional relevance. In particular, it is unknown whether the motif is relevant for disulfide formation and to what extent it would affect the profilins’ structural, functional and immunological properties. Here we present crystal structures of Bet v 2 in the reduced and the oxidized state, i.e., without and with a disulfide bridge. Despite overall structural similarity, the two structures distinctly differ at their termini which are stabilized to each other in the oxidized, i.e., disulfide-linked state. These structural differences translate into differences in their proteolytic resistance. Whereas the oxidized Bet v 2 is rather resistant towards the endolysosomal protease cathepsin S, it is rapidly degraded in the reduced form. By contrast, both Bet v 2 forms exhibit similar immunological properties as evidenced by their binding to IgE antibodies from birch pollen allergic patients and by their ability to trigger histamine release in a humanized rat basophilic leukemia cells (RBL) assay, independent of the presence or absence of the disulfide bridge. Taken together our findings suggest that the oxidized Bet v 2 conformation should be the relevant species, with a much longer retention time to trigger immune responses.

Background Performing multiple high-intensity interval training (HIIT) sessions in a compressed period of time (approximately 7–14 days) is called a HIIT shock microcycle (SM) and promises a rapid increase in endurance performance. However, the efficacy of HIIT-SM, as well as knowledge about optimal training volumes during a SM in the endurance-trained population have not been adequately investigated. This study aims to examine the effects of two different types of HIIT-SM (with or without additional low-intensity training (LIT)) compared to a control group (CG) on key endurance performance variables. Moreover, participants are closely monitored for stress, fatigue, recovery, and sleep before, during and after the intervention using innovative biomarkers, questionnaires, and wearable devices. Methods This is a study protocol of a randomized controlled trial that includes the results of a pilot participant. Thirty-six endurance trained athletes will be recruited and randomly assigned to either a HIIT-SM (HSM) group, HIIT-SM with additional LIT (HSM + LIT) group or a CG. All participants will be monitored before (9 days), during (7 days), and after (14 days) a 7-day intervention, for a total of 30 days. Participants in both intervention groups will complete 10 HIIT sessions over 7 consecutive days, with an additional 30 min of LIT in the HSM + LIT group. HIIT sessions consist of aerobic HIIT, i.e., 5 × 4 min at 90–95% of maximal heart rate interspersed by recovery periods of 2.5 min. To determine the effects of the intervention, physiological exercise testing, and a 5 km time trial will be conducted before and after the intervention. Results The feasibility study indicates good adherence and performance improvement of the pilot participant. Load monitoring tools, i.e., biomarkers and questionnaires showed increased values during the intervention period, indicating sensitive variables. Conclusion This study will be the first to examine the effects of different total training volumes of HIIT-SM, especially the combination of LIT and HIIT in the HSM + LIT group. In addition, different assessments to monitor the athletes' load during such an exhaustive training period will allow the identification of load monitoring tools such as innovative biomarkers, questionnaires, and wearable technology. Trial Registration : clinicaltrials.gov, NCT05067426. Registered 05 October 2021—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05067426 . Protocol Version Issue date: 1 Dec 2021. Original protocol. Authors: TLS, NH.