Grass Pollen Allergens Research Articles

Sensitizations to pollen differ between Central European and Sub-Saharan African atopic dermatitis patients

Summary Background Atopic dermatitis (AD) is often associated with allergic comorbidities, such as allergic asthma or allergic rhinoconjunctivitis (ARC). Sensitizations to pollen can directly impact AD, as patients can experience exacerbation during pollen season. This study aims to gain more insights into the pollen sensitization patterns of AD patients in Central Europe compared with sub-Saharan Africa (SSA). Methods We performed a case–control study involving a total of 90 participants: 20 AD patients and 10 healthy controls (HC) each from Switzerland (CH), Tanzania (TZ), and Madagascar (MD). We collected clinical data and serum samples and performed a multiplex IgE test (ALEX2 Allergy Explorer, MacroArray Diagnostics, Vienna, Austria). Results The prevalence of ARC and asthma in AD patients was similar in all countries (ARC: 60% TZ, 70% CH, 75% MD; asthma: 25% TZ, 30% CH, 20% MD). Total IgE levels were significantly higher in both SSA HC populations compared with the Swiss HC. The analysis of specific IgE levels revealed major differences in sensitization patterns between Africa and Europe, especially regarding grass pollen allergens. Swiss AD patients were sensitized to various grass pollen such as Bahia grass, Bermuda grass, common reed, perennial ryegrass, rye, and timothy grass. However, these allergens were irrelevant in the SSA population: no AD patient or HC subject was sensitized to the tested grass pollen. Conclusion The considerably different sensitization patterns between European and SSA AD patients warrant the development of allergy testing and desensitization therapies tailored to the African setting. Therefore, there is a need to characterize local pollen types and counts.

Dupilumab Improves Clinical Symptoms and Biomarkers in Comorbid Seasonal Timothy Grass Pollen Allergic Rhinitis in Patients With CRSwNP.

Dupilumab Improves Clinical Symptoms and Biomarkers in Comorbid Seasonal Timothy Grass Pollen Allergic Rhinitis in Patients With CRSwNP.

Identification of cross-reactive IgE-binding proteins from Philippine allergenic grass pollen extracts.

Respiratory allergies are one of the most common allergic diseases that affect Filipinos. Grass pollen accounts for the majority of the outdoor allergens triggering these respiratory allergies. Cross-reactivity among the Philippine grass pollen grains has not been extensively studied. This study aims to investigate the cross-reactivity of our local grasses and identify the cross-reactive allergens. Grass pollen grains were collected and processed into crude allergenic extracts. The IgE-reactivity of these crude allergenic pollen extracts was studied using sera from patients who tested positive for the mentioned extracts. The proteins from the immunoblots of cross-reactive pollen allergen extracts were sequenced and identified. Allergenic pollen proteins were identified as cross-reactive among the grass pollen extracts. Four of these have not been listed yet as grass allergens in the World Health Organization/International Union of Immunological Societies allergen nomenclature database. Local grass pollen allergens are cross-reactive with probable new allergens identified.

Adoptive Cell Therapy in Mice Sensitized to a Grass Pollen Allergen.

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5+ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5+ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5+ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy.

The experience with subcutaneous allergen-specific immunotherapy with pollen allergens in patients with atopic dermatitis

BACKGROUND: Atopic dermatitis is a chronic inflammatory skin disease, the key pathogenetic mechanisms of which include activation of T2-immune inflammation, IgE-specific sensitization to various allergens, and disruptions of epidermal barrier functions. Allergen-specific immunotherapy is the only pathogenetic treatment method that can prevent the progression of the disease and the development of the atopic march. Currently, clinical experience has been accumulated in using allergen-specific immunotherapy for atopic dermatitis, yet the existing evidence base does not allow for definitive conclusions regarding the indications for conducting allergen-specific immunotherapy, especially in atopic dermatitis associated with pollen allergy.
 AIMS: Determination of the clinical significance of sensitization to pollen allergens in atopic dermatitis, determination of indications for allergen-specific immunotherapy with pollen allergens and assessment of the effectiveness and safety of this method in atopic dermatitis patients.
 MATERIALS AND METHODS: A comparative controlled study to evaluate the effectiveness of subcutaneous allergen-specific immunotherapy with pollen allergens of birch or cereal grasses in 78 adult atopic dermatitis patients was conducted. A single course of subcutaneous allergen-specific immunotherapy with water-salt solutions of tree pollen allergens was administered to 33 patients, and grass pollen allergens were administered to 21 patients with atopic dermatitis. The comparison group ― patients with atopic dermatitis who received standard therapy for the disease without allergen-specific immunotherapy ― 24 patients. The primary endpoints of the study were to evaluate the effectiveness of therapy based on the reduction in the severity of atopic dermatitis symptoms, based on the absolute and relative number of patients who achieved a reduction in SCORAD score by 75% and IGA 1/0.
 RESULTS: In patients receiving one course of subcutaneous allergen-specific immunotherapy with tree pollen allergens, SCORAD 50 achieved 26, 33, 47% and SCORAD 75 achieved 10, 20 and 33% after 16, 32 and 52 weeks, respectively. In patients who received one course of subcutaneous allergen-specific immunotherapy with grass pollen allergens, SCORAD 50 reached 31, 40, 58% and SCORAD 75 ― 14, 28 and 43% after 16, 32 and 52 weeks, respectively. In the comparison group, the number of such patients was significantly smaller: SCORAD 50 reached 24, 32, 37% and SCORAD 75 ― 13, 22 and 26% after 16, 32 and 52 weeks, respectively. Similar results were obtained when studying the IGA. In all study groups, an improvement in the quality of life of patients was noted, which was reflected in a decrease in the DLQI index by 4 or more points in 12, 25 and 37% of patients receiving one course of subcutaneous allergen-specific immunotherapy with tree pollen allergens, in 14, 28 and 43% in patients after 16, 32 and 52 weeks, respectively. However, the number of such patients in the comparison group was significantly lower: 13, 22 and 26%, respectively.
 CONCLUSION: Allergen-specific immunotherapy is an effective way to treat atopic dermatitis patients, provided there is proven sensitization to pollen allergens.

Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

A Case-Control Study Comparing the General Characteristics of Patients with Symptomatic Dermographism and Chronic Spontaneous Urticaria: Is Atopy a Risk Factor for Symptomatic Dermographism?

Introduction: Symptomatic dermographism (SDerm) is the most common chronic inducible urticaria (CIndU) subtype. There is still limited information in the literature about clinical features, triggering factors, and accompanying comorbidities of SDerm. The aim of this study was to compare the clinical features and laboratory data of patients with SDerm and chronic spontaneous urticaria (CSU). Methods: The clinical features and laboratory data of patients with SDerm and CSU were compared retrospectively. The laboratory data and general characteristic features of the patients were obtained from the medical records. Results: The study included a total of 361 patients (CSU: 220, SDerm: 141). The rates of asthma (odds ratio [OR]: 1.79, p = 0.036), allergic rhinitis (OR: 6.03, p < 0.001), and thyroid disease (OR: 1.78, p = 0.039) were higher in patients with SDerm. The disease duration (median 12 months, p < 0.001) and regular antihistamine use (OR: 0.31, p < 0.001) were lower in patients with SDerm. Total IgE level (median: 193, p < 0.001), thyroid antibody positivity (OR: 1.93, p = 0.039), and atopy (OR: 8.81, p < 0.001) were higher in patients with SDerm. Dermatophagoides pteronyssinus (OR: 17.72, p < 0.001), Dermatophagoides farinae (OR: 17.20, p < 0.001), grass pollen (OR: 2.50, p < 0.026), cat epithelium (OR: 3.68, p < 0.023), and cockroach (OR: 4.93, p < 0.009) allergen positivity rates were higher in patients with SDerm. Conclusion: Atopic diseases such as asthma and allergic rhinitis and the sensitization rate to aeroallergens seem to be higher in patients with SDerm than in patients with CSU. The results of this study should be supported by multicenter studies of patients from different geographical regions.

Oligomerization and tyrosine nitration enhance the allergenic potential of the birch and grass pollen allergens Bet v 1 and Phl p 5.

Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide (environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied.

Adoptive transfer of allergen-expressing B cells prevents IgE-mediated allergy.

Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells) to induce allergen-specific tolerance in naive mice and identified CD19+ B cells as promising candidates for allergen-specific cell therapy. For this purpose, CD19+ B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing. The transfer of purified Phl p 5-expressing CD19+ B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19+ B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice. Thus, we demonstrated that the transfer of Phl p 5-expressing CD19+ B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans.

Immunothérapie allergénique aux pollens de graminées : profil de sécurité et d’efficacité

IntroductionRespiratory allergies to grass pollens are very widespread in the general population and the treatment is generally symptomatic which sometimes remains insufficient. Grass pollen allergen immunotherapy is currently the only etiological treatment of seasonal allergic rhinitis apart from allergenic eviction. AimThe aim was to study the effectiveness of sublingual ITA tablets with 5-grasses on allergic disease and describe the adverse effects after one year of treatment. MethodsWe conducted a prospective, descriptive and evaluative study including 40 patients suffering from grass pollen allergic rhinitis. A grass pollen sublingual allergen immunotherapy was received and patients were followed during a one-year treatment to study the efficacy and the safety profile. ResultsThe mean age was 30.9±12.25 years. The sex ratio (M/F) was 0.48. Allergic conjunctivitis was associated in 70% and allergic asthma was associated in 48% of cases. Allergen immunotherapy side effects were mild, consisting of local side effects. No severe side effects were observed. A decrease in allergic rhinitis symptoms was observed in 83% with improvement in medication score in 65%. All patients noted a decrease in conjunctivitis symptoms with a total regression in 54% of cases. A better asthma control was noted in 33%. Among the 19 patients suffering from asthma, we could decrease the drug use in 63% of cases during the first year of treatment. An improvement of the quality of life was observed in 78% of cases. Allergen immunotherapy was significantly more efficient in allergic rhinitis and conjunctivitis with patients under 30years old. ConclusionGrass pollen allergen immunotherapy leads safely to the regression of allergy symptoms and improves the therapeutic load and the quality of life.

In Silico Design of a New Epitope-Based Vaccine against Grass Group 1 Allergens.

Allergic diseases are a global public health problem that affects up to 30% of the population in industrialized societies. More than 40% of allergic patients suffer from grass pollen allergy. Grass pollen allergens of group 1 and group 5 are the major allergens, since they induce allergic reactions in patients at high rates. In this study, we used immunoinformatic approaches to design an effective epitope-based vaccine against the grass group 1 allergens. After the alignment of all known pollen T-cell and B-cell epitopes from pollen allergens available in the public databases, the epitope GTKSEVEDVIPEGWKADTSY was identified as the most suitable for further analyses. The target sequence was subjected to immunoinformatics analyses to predict antigenic T-cell and B-cell epitopes. Population coverage analysis was performed for CD8+ and CD4+ T-cell epitopes. The selected T-cell epitopes (VEDVIPEGW and TKSEVEDVIPEGWKA) covered 78.87% and 98.20% of the global population and 84.57% and 99.86% of the population of Europe. Selected CD8+, CD4+ T-cell and B-cell epitopes have been validated by molecular docking analysis. CD8+ and CD4+ T-cell epitopes showed a very strong binding affinity to major histocompatibility complex (MHC) class I (MHC I) molecules and MHC class II (MHC II) molecules with global energy scores of -72.1 kcal/mol and -89.59 kcal/mol, respectively. The human IgE-Fc (PDB ID 4J4P) showed a lower affinity with B-cell epitope (ΔG = -34.4 kcal/mol), while the Phl p 2-specific human IgE Fab (PDB ID 2VXQ) had the lowest binding with the B-cell epitope (ΔG = -29.9 kcal/mol). Our immunoinformatics results demonstrated that the peptide GTKSEVEDVIPEGWKADTSY could stimulate the immune system and we performed ex vivo tests showed that the investigated epitope activates T cells isolated from patients with grass pollen allergy, but it is not recognized by IgE antibodies specific for grass pollen allergens. This confirms the importance of such studies to establish universal epitopes to serve as a basis for developing an effective vaccine against a particular group of allergens. Further in vivo studies are needed to validate the effectiveness of such a vaccine against grass pollen allergens.

Assessing IgE sensitization profiles to birch and timothy grass pollen allergens in birch pollen allergic blood donors using an oligoplex specific IgE assay

IgE sensitization profiles to single birch allergens in birch-sensitized patients differ among European countries. The aim of this study was to determine the distribution of specific IgE antibodies to major and minor birch pollen allergens in a population of allergic Norwegian individuals by using a birch allergic blood donor population as a surrogate sample. Sixty blood donors were recruited and sampled based on birch allergy symptoms such as rhinitis, rhinoconjunctivitis and/or mild asthma in previous seasons. All sera were collected before start of the pollen season and tested using a line blot assay (Euroimmun AG, Lübeck, Germany) for IgE to birch and timothy pollen. Both extracts, single allergens, and cross-reacting carbohydrate determinants (CCD) were analysed. Only donors with specific IgE to birch and/or timothy grass were further evaluated. Specific IgE to birch pollen extract was found in 52 sera, and sensitization to timothy grass in 40 sera. Specific IgE to Bet v 1 was predominant in contrast to Bet v 4 which was absent. However, sensitization to the minor allergens Bet v 2 and 6 was always found together with high levels of IgE to Bet v 1. Subjects sensitized to the profilin Bet v 2 from birch were also sensitized to Phl p 12 from timothy grass. In conclusion, there was predominantly Bet v 1 sensitization in this cohort and low sensitization to minor allergens and cross-reactive allergens (Bet v 2, Bet v 4, Phl p 7 and Phl p 12).

A molecular sensitization map of European children reveals exposome- and climate-dependent sensitization profiles.

Understanding differences in sensitization profiles at the molecular allergen level is important for diagnosis, personalized treatment and prevention strategies in allergy. Immunoglobulin E (IgE) sensitization profiles were determined in more than 2800 sera from children in nine population-based cohorts in different geographical regions of Europe; north [BAMSE (Sweden), ECA (Norway)], west/central [PIAMA (the Netherlands), BiB (the United Kingdom), GINIplus (Germany)], and south [INMA Sabadell and Gipuzkoa (Spain) and ROBBIC Rome and Bologna (Italy)] using the MeDALL-allergen chip. Sensitization to grass pollen allergen, Phl p 1, and to major cat allergen, Fel d 1, dominated in most European regions whereas sensitization to house dust mite allergens Der p 1, 2 and 23 varied considerably between regions and were lowest in the north. Less than half of children from Sabadell which has a hot and dry climate were sensitized to respiratory allergens, in particular house dust mite allergens as compared to Gipuzkoa nearby with a more humid climate. Peanut allergen Ara h 1 was the most frequently recognized class 1 food allergen in Northern/Western Europe, while the fruit allergens Pru p 3, Act d 1 and 2 were prominent in Southern and Western/Central Europe. Ves v 5-sensitization dominated in North and West/Central Europe. We show regional, exposome- and climate-dependent differences in molecular IgE-reactivity profiles in Northern, Western/Central and Southern Europe which may form a molecular basis for precision medicine-based approaches for treatment and prevention of allergy.

Clonal evolution and stereotyped sequences of human IgE lineages in aeroallergen-specific immunotherapy

Allergic disease reflects specific inflammatory processes initiated by interaction between allergen and allergen-specific IgE. Specific immunotherapy (SIT) is an effective long-term treatment option, but the mechanisms by which SIT provides desensitization are not well understood. Our aim was to characterize IgE sequences expressed by allergen-specific B cells over a 3-year longitudinal study of patients with aeroallergies who were undergoing SIT. Allergen-specific IgE-expressing clones were identified by using combinatorial single-chain variable fragment libraries and tracked in PBMCs and nasal biopsy samples over a 3-year period with antibody gene repertoire sequencing. The characteristics of private IgE-expressing clones were compared with those of stereotyped or "public" IgE responses to the grass pollen allergen Phleum pratense (Phl p) 2. Members of the same allergen-specific IgE lineages were observed in nasal biopsy samples and blood, and lineages detected at baseline persisted in blood and nasal biopsy samples after 3 years of SIT, including B cells that express IgE. Evidence of progressive class switch recombination to IgG subclasses was observed after 3 years of SIT. Acommon stereotyped Phl p 2-specific antibody heavy chain sequence was detected in multiple donors. The amino acid residues enriched in IgE-stereotyped sequences from seropositive donors were analyzed with machine learning and k-mer motif discovery. Stereotyped IgE sequences had lower overall rates of somatic hypermutation and antigen selection than did single-chain variable fragment-derived allergen-specific sequences or IgE sequences of unknown specificity. Longitudinal tracking of rare circulating and tissue-resident allergen-specific IgE+ clones demonstrates persistence of allergen-specific IgE+ clones, progressive class switch recombination to IgG subtypes, and distinct maturation of a stereotyped Phl p 2 clonotype.

Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5.

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO-) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO- and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO--induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.

Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Single-cell RNA-Seq identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Single-cell RNA-Seq (scRNA-seq) provides a novel approach to identify novel tolerogenic molecular pathways induced by allergen immunotherapy. We sought to investigate the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized grass pollen extract for allergen immunotherapy.

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy.

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Immunologic mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded suboptimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA sequencing technology offers a bridge to this gap in knowledge. We sought to identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense (Phl p) extract. The molecular mechanisms underlying native Phl p, depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated by single-cell RNA sequencing. Allergen-specific TH2A, T follicular helper (Tfh), and IL-10+ regulatory B cells were quantified by flow cytometry in peripheral blood mononuclear cells from 16 grass pollen-allergic and 8 nonatopic control subjects. The ability of Phl p, DPG-Phl p, and DPG-POL-Phl p to elicit FcεRI- and FcεRII-mediated IgE responses was measured by basophil activation test and IgE-facilitated allergen binding assay. Analysis revealed that DPG-POL-Phl p downregulated genes associated with TH2 signaling, induced functional regulatory T cells exhibiting immunosuppressive roles through CD52 and Siglec-10, modulated genes encoding immunoproteasome that dysregulate the processing and presentation of antigens to T cells and promoted a shift from IgE toward an IgA1 and IgG responses. In grass pollen-allergic subjects, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of TH2A, IL-4+ Tfh and IL-21+ Tfh cells while being the most prominent at inducing IL-10+CD19+CD5hi and IL-10+CD19+CD5hiCD38intCD24int regulatory B-cell subsets compared to Phl p (all P< .05). Furthermore, DPG-POL-Phl p demonstrated a hypoallergenic profile through basophil activation and histamine release compared to Phl p (31.54-fold, P< .001). Single-cell RNA sequencing provides an in-depth resolution of the mechanisms underlying the tolerogenic profile of DPG-POL-Phl p.

BAYESIAN NETWORK ANALYSIS INDICATES A HIGH PROBABILITY OF SIMULTANEOUS SENSITIZATION TO MAJOR GRASS ALLERGENS

<h3>Introduction</h3> Grass pollen allergy impacts many patients characterized by high allergen cross-reactivity. The primary elicitor of grass allergy is Phl p 1. However, IgE to Phl p 5 or Phl p 2 also serve as markers of true grass pollen sensitization. Understanding the patterns of sensitization to grasses in certain population remains important for accurate prevention and treatment of allergy to Poaceae pollen. <h3>Methods</h3> Data obtained by multiplex allergy testing Alex² was collected from 20,333 patients. Bayesian Network analysis was used to build probabilistic patterns of patient sensitization to majpr grass pollen allergens. <h3>Results</h3> Grass pollen sensitive individuals constituted 6170 patients or 30.34 % of the entire database; 3935 (19.35 %) were sensitive to Phl p 1; 3460 (17.02 %) – to Lol p 1; 2842 (13.98 %) – to Cyn d 1; and 1772 (8.71 %) – to Phl p 2. Bayesian Network analysis indicated that sensitization to Phl p 1 was associated with sensitivity to Cyn d 1, Lol p 1, Phl p 2 and Sec c_pollen with 94.70 % probability. Sensitization to Lol p 1 was connected with sensitivity to Cyn d 1, Pas n extract, Phl p 2, Phl p 5 with 91.96 % probability. Cyn d 1 sensitization could be seen together with Cyn d, Sec c_pollen, Phr c extracts and Phl p 6 sensitization in 86.58 % of cases. <h3>Conclusion</h3> Bayesian Network analysis suggests that group I allergens are highly cross-reactive and can be a good target for AIT in the Ukrainian population sensitive to grass pollen.