Structural Similarities, in Relation with the Cross-Reactivity, of Hymenoptera Allergenic Dipeptidyl Peptidases IV-An Overall Comparison Including a New Dipeptidyl Peptidase IV Sequence from Vespa velutina.

(1) Background: Dipeptidyl Peptidases IV (DPPIVs), present in many organisms, are minor components in the venoms of Hymenoptera, where they have been identified as cross-reactive allergenic molecules. Considering that the structure of homologous DPPIVs is well characterized, we aimed to explain which regions have higher similarity among these proteins and present a comparison among them, including a new Vespa velutina DPPIV sequence. Moreover, two cases of sensitization to DPPIVs in wasp- and honeybee-sensitized patients are presented. (2) Methods: Proteomic analyses have been performed on the venom of the Asian hornet Vespa velutina to demonstrate the sequence of its DPPIV (allergen named Vesp v 3, with sequence accession number P0DRB8, and with the proteomic data available via ProteomeXchange with the identifier PXD046030). A comparison performed through their alignments and analysis of the three-dimensional structure showed a region with higher similarity among Hymenoptera DPPIVs. Additionally, ImmunoCAP™ determinations (including specific inhibition experiments), as well as IgE immunoblotting, are performed to demonstrate the allergenicity of Api m 5 and Ves v 3. (3) Results and Conclusions: The data presented demonstrate that the similarities among Hymenoptera DPPIVs are most likely localized at the C-terminal region of these enzymes. In addition, a higher similarity of the Vespa/Vespula DPPIVs is shown. The clinical cases analyzed demonstrated the allergenicity of Api m 5 and Ves v 3 in the sera of the allergic patients, as well as the presence of this minor component in the preparations used in venom immunotherapy.

Supplementation with inulin-type fructans affects gut microbiota and attenuates some of the cardiometabolic benefits of a plant-based diet in individuals with overweight or obesity.

Background: The gut microbiota has emerged as a potential therapeutic target to improve the management of obesity and its comorbidities. Objective: We investigated the impact of a high fiber (∼38 g/d) plant-based diet, consumed ad libitum, with or without added inulin-type fructans (ITF), on the gut microbiota composition and cardiometabolic outcomes in subjects with obesity. We also tested if baseline Prevotella/Bacteroides (P/B) ratio predicts weight loss outcomes. Methods: This is a secondary exploratory analysis from the PREVENTOMICS study, in which 100 subjects (82 completers) aged 18-65 years with body mass index 27-40 kg/m2 were randomized to 10 weeks of double-blinded treatment with a personalized or a generic plant-based diet. Changes from baseline to end-of-trial in gut microbiota composition (16S rRNA gene amplicon sequencing), body composition, cardiometabolic health and inflammatory markers were evaluated in the whole cohort (n = 82), and also compared in the subgroup of subjects who were supplemented with an additional 20 g/d ITF-prebiotics (n = 21) or their controls (n = 22). Results: In response to the plant-based diet, all subjects lost weight (-3.2 [95% CI -3.9, -2.5] kg) and experienced significant improvements in body composition and cardiometabolic health indices. Addition of ITF to the plant-based diet reduced microbial diversity (Shannon index) and selectively increased Bifidobacterium and Faecalibacterium (q < 0.05). The change in the latter was significantly associated with higher values of insulin and HOMA-IR and lower HDL cholesterol. In addition, the LDL:HDL ratio and the concentrations of IL-10, MCP-1 and TNFα were significantly elevated in the ITF-subgroup. There was no relationship between baseline P/B ratio and changes in body weight (r = -0.07, p = 0.53). Conclusion: A plant-based diet consumed ad libitum modestly decreases body weight and has multiple health benefits in individuals with obesity. Addition of ITF-prebiotics on top this naturally fiber-rich background selectively changes gut microbiota composition and attenuates some of the realized cardiometabolic benefits. Clinical trial registration: [https://clinicaltrials.gov/ct2/show/NCT04590989], identifier [NCT04590989].

Personal diet-microbiota interactions and weight loss.

The aim of this review is to provide an overview of how person-specific interactions between diet and the gut microbiota could play a role in affecting diet-induced weight loss responses. The highly person-specific gut microbiota, which is shaped by our diet, secretes digestive enzymes and molecules that affect digestion in the colon. Therefore, weight loss responses could in part depend on personal colonic fermentation responses, which affect energy extraction of food and production of microbial metabolites, such as short-chain fatty acids (SCFAs), which exert various effects on host metabolism. Colonic fermentation is the net result of the complex interplay between availability of dietary substrates, the functional capacity of the gut microbiome and environmental (abiotic) factors in the gut such as pH and transit time. While animal studies have demonstrated that the gut microbiota can causally affect obesity, causal and mechanistic evidence from human studies is still largely lacking. However, recent human studies have proposed that the baseline gut microbiota composition may predict diet-induced weight loss-responses. In particular, individuals characterised by high relative abundance of Prevotella have been found to lose more weight on diets rich in dietary fibre compared to individuals with low Prevotella abundance. Although harnessing of personal diet-microbiota interactions holds promise for more personalised nutrition and obesity management strategies to improve human health, there is currently insufficient evidence to unequivocally link the gut microbiota and weight loss in human subjects. To move the field forward, a greater understanding of the mechanistic underpinnings of personal diet-microbiota interactions is needed.

Allergen-specific IgG+ memory B cells are temporally linked to IgE memory responses.

BACKGROUND: IgE is the least abundant immunoglobulin and tightly regulated, and IgE-producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood. OBJECTIVE: The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated. METHODS: In a randomized double-blind, placebo-controlled, time course SLIT study, PBMCs and nasal biopsy samples were collected from 40 adults with seasonal allergic rhinitis at baseline and at 4, 8, 16, 28, and 52 weeks. RNA was extracted from PBMCs, sorted B cells, and nasal biopsy samples for heavy chain variable gene repertoire sequencing. Moreover, mAbs were derived from single B-cell transcriptomes. RESULTS: Combining heavy chain variable gene repertoire sequencing and single-cell transcriptomics yielded direct evidence of a parallel boost of 2 clonally and functionally related B-cell subsets of short-lived IgE+ plasmablasts and IgG+ memory B cells. Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgGE repertoires. These were extensively mutated and appeared relatively stable as per heavy chain isotype, somatic hypermutations, and clonal composition. Single IgGE+ memory B-cell and IgE+ preplasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and able to elicit basophil activation at very low allergen concentrations. CONCLUSION: For the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG+ memory B cells. These cells rapidly switch isotype, expand into short-lived IgE+ plasmablasts, and serve as a potential target for therapeutic intervention.

Diverse and highly cross-reactive T-cell responses in ragweed allergic patients independent of geographical region.

BACKGROUND: Ragweed frequently causes seasonal allergies in North America and Europe. In the United States, several related ragweed species with diverse geographical distribution cause allergic symptoms. Cross-reactivity towards related ragweed species of IgE and treatment-induced IgG4 has been demonstrated previously. However, less is known about the underlying T-cell cross-reactivity. METHODS: The allergen content of ragweed extracts was determined by mass spectrometry and related to T-cell epitopes of Amb a allergens (group 1, 3, 4, 5, 8 and 11) in 20 American ragweed allergic patients determined by FluoroSpot and proliferation assays. T-cell responses to 50 frequently recognized Amb a-derived T-cell epitopes and homologous peptides from western ragweed (Amb p), giant ragweed (Amb t) and mugwort (Art v) were investigated in an additional 11 American and 14 Slovakian ragweed allergic donors. RESULTS: Ragweed extracts contained all known allergens and isoallergens thereof. Donor T-cell responses were diverse and directed against all Amb a 1 isoallergens and to most minor allergens investigated. Similar response patterns were seen in American and Slovakia donors. Several epitopes were cross-reactive between isoallergens and ragweed species, some even including mugwort. T-cell cross-reactivity generally correlated with allergen sequence homology. CONCLUSION: T-cell epitopes of multiple allergens/isoallergens are involved in the diverse T-cell responses in ragweed allergic individuals. T-cell lines were highly cross-reactive to epitopes of related ragweed species without any apparent geographical response bias. These data support that different ragweed species can be considered an allergen homology group with Amb a as the representative species regarding diagnosis as well as allergy immunotherapy.

Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity.

BACKGROUND: House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking. OBJECTIVE: To comprehensively analyse the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function. METHODS: Proteomic analysis (liquid chromatography-tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity, and putative function analyses were performed in silico according to Gene Ontology annotations. RESULTS: Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T cell response, underlining the heterogeneity of T cell responses to HDM allergens. CONCLUSIONS AND CLINICAL RELEVANCE: Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM.

Short ragweeds is highly cross-reactive with other ragweeds.

BACKGROUND: The most widespread ragweed (Ambrosia) species in North America are short ragweed (Ambrosia artemisiifolia; Amb a), giant ragweed (Ambrosia trifida; Amb t), and western ragweed (Ambrosia psilostachya; Amb p). Varied geographic distributions of ragweed species raise questions regarding the need for ragweed species-specific allergen immunotherapy. OBJECTIVE: To determine allergenic cross-reactivity among ragweed species by immunologic analyses of sera from subjects allergic to ragweed from North America and Europe. METHODS: Sera were collected from 452 subjects allergic to ragweed who participated in Amb a sublingual immunotherapy tablet clinical trials. All subjects had positive skin prick test and serum IgE against Amb a. Ragweed-specific IgE (pre treatment) and IgG4 (post treatment) were measured by ImmunoCAP. IgE inhibition studies among Amb a, Amb t, and Amb p were conducted. Using pooled sera from another ragweed-allergic population, IgE inhibition studies of 7 less widespread Ambrosia species also were conducted. RESULTS: A strong correlation between Amb a vs Amb p and Amb t serum IgE levels was observed. In the vast majority of pretreatment sera, Amb a inhibited Amb a, Amb p, and Amb t IgE reactivity by more than 90%. Strong correlations were observed between Amb a vs Amb p and Amb t post-treatment IgG4 levels. In pooled sera, Amb a extract inhibited the binding of serum IgE to all 10 ragweed species by 98%-100%. CONCLUSION: In a population of subjects allergic to Amb a, substantial allergenic cross-reactivity among Amb a, Amb p, and Amb t was demonstrated. These in vitro data suggest that an Amb a-based single-species ragweed allergen immunotherapy may be therapeutically active in patients exposed to diverse ragweed pollens. TRIAL REGISTRY: Clinicaltrials.gov, NCT00770315, NCT00783198, and NCT00330083.

Effect of mutations on the thermostability of Aspergillus aculeatus ß-1,4-galactanase.

New variants of ß-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of ß-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm ) by differential scanning calorimetry. We found that the Tm is elevated by 1.1 °C for G306A, slightly increased (in the range of 0.34 to 0.65 °C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.

Facilitated antigen presentation and its inhibition by blocking IgG antibodies depends on IgE repertoire complexity.

BACKGROUND: The antibody repertoires of allergic subjects are characterized by the presence of allergen-specific IgE antibodies. We have previously shown that the composition of the IgE repertoire is critical for allergen-mediated activation of human effector cells. Activation of CD4(+) T cells in allergic subjects is highly potentiated by the process of facilitated antigen presentation (FAP), in which allergen in complex with IgE is taken up by B cells through the low-affinity IgE receptor CD23 and presented to T cells. OBJECTIVE: We sought to investigate the influence of IgE repertoire complexity on the formation of IgE/allergen/CD23 complexes on B cells and subsequent T-cell activation. METHODS: Using defined allergen-specific recombinant IgE and IgG antibodies, we investigated the influence of individual IgE affinity, IgE clonality, specific IgE concentration, and the ratio between IgE specificities on IgE/allergen/CD23 complex formation in vitro. RESULTS: Although IgE affinity is an important factor, IgE clonality seems to be governing complex formation, especially with medium- and low-affinity IgE antibodies. We demonstrate that differences in allergen-specific IgE affinity correlate with the efficiency of subsequent T-cell activation. In addition, we show that the complexity of an IgE repertoire also affects the ability of allergen-specific IgG antibodies to block FAP. CONCLUSION: The composition of allergen-specific IgE repertoires in individual patients, especially with respect to IgE clonality, might play an important role in the manifestation of allergic disease not only for the immediate allergic reaction through activation of basophils and mast cells but also for the exacerbation of allergic inflammation through recurring activation of allergen-specific T cells by FAP.

Isoallergen variations contribute to the overall complexity of effector cell degranulation: effect mediated through differentiated IgE affinity.

Most allergens exist in several variants (isoallergens), each of which may be recognized differently by patient IgE. We have previously shown that several properties of the IgE repertoire, including IgE affinity and IgE clonality, are important factors determining degranulation responses of effector cells involved in type I allergic reactions. However, less is known about how the repertoire of naturally occurring isoallergens may affect this response. Thus, in this study, we investigated how individual rIgE Ab clones derived from a human subject are able to distinguish among variants of Der p 2 isoallergens and assessed the impact on basophil degranulation. Biacore analyses showed that individual rIgE clones cloned from an individual allergic to house dust mites recognized Der p 2 with binding affinities varying up to 100-fold between different Der p 2 isoforms. In a well-defined biological system consisting of human basophils sensitized with low rIgE clonality, degranulation responses were directly related to rIgE affinity toward particular rDer p 2 isoallergens. However, basophils sensitized with polyclonal patients' sera showed no differences in degranulation responses toward the different rDer p 2 isoallergens. In conclusion, our study shows that individual IgE Abs are able to bind single allergens with a broad range of affinities due to natural isoallergen variations, contributing further to the overall complexity of IgE-allergen interactions at the effector cell surface, which is, however, blurred by the polyclonal nature of patients' IgE repertoires.