Advances and novel developments in molecular allergology.

The continuous search for new allergens and the design of allergen derivatives improves the understanding of their allergenicity and aids the design of novel diagnostic and immunotherapy approaches. This article discusses the recent developments in allergen and epitope discovery, allergy diagnostics and immunotherapy. Structural information is crucial for the elucidation of cross-reactivity of marker allergens such as the walnut Jug r 6 or that of nonhomologous allergens, as shown for the peanut allergens Ara h 1 and 2. High-throughput sequencing, liposomal nanoallergen display, bead-based assays, and protein chimeras have been used in epitope discovery. The binding of natural ligands by the birch pollen allergen Bet v 1 or the mold allergen Alt a 1 increased the stability of these allergens, which is directly linked to their allergenicity. We also report recent findings on the use of component-resolved approaches, basophil activation test, and novel technologies for improvement of diagnostics. New strategies in allergen-specific immunotherapy have also emerged, such as the use of virus-like particles, biologics or novel adjuvants. The identification of dectin-1 as a key player in allergy to tropomyosins and the formyl peptide receptor 3 in allergy to lipocalins are outstanding examples of research into the mechanism of allergic sensitization.

Increased antiviral response in circulating lymphocytes from hypogammaglobulinemia patients.

BACKGROUND: B cells play a crucial role during rhinovirus (RV) infections by production of virus-neutralizing antibodies. A main feature of common variable immunodeficiency (CVID) is hypogammaglobulinemia (HG). HG patients have severely reduced levels of antibody-producing B cells and suffer from prolonged virus infections. Here, we addressed whether antiviral response of peripheral blood lymphocytes differs between HG patients and healthy individuals during natural RV infection. METHODS: Using fluorescence-activated cell sorting, B-cell subsets were analyzed. Simultaneously, CD19 + B cells, CD14 + monocytes, and CD3 + T cells were sorted from frozen peripheral blood mononuclear cells from 11 RV-infected hypogammaglobulinemia patients, 7 RV-infected control subjects, and 14 noninfected control subjects. Real-time PCR was used to study expression of antiviral genes. A pan-RV PCR was used to detect RV genome in all samples. RESULTS: In HG patients, total B-cell numbers, as well as IgA + and IgG + switched memory B cells, were reduced while naïve B cells and T cells were increased. STAT1 expression was increased in HG patients compared to controls in all lymphocyte subsets analyzed. The expression of antiviral genes IFITM1 and MX1 correlated with STAT1 expression in B cells and monocytes. RV RNA was found in 88.9% of monocytes from infected HG patients, 85.7% of monocytes from infected controls, and 7.1% of monocytes from uninfected controls. CONCLUSIONS: We demonstrate an increased antiviral response in B cells and monocytes in HG patients and their correlation with STAT1 expression. Monocytes of infected HG patients and infected non-HG controls carry RV RNA.

Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein.

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein⁻lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

Bacillus thuringiensis Cyt2Aa2 binding on lipid/cholesterol bilayer depends on protein concentration and time.

Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 µg/ml and 100 µg/ml after 30 min. At lower concentrations, 10 µg/ml and 17.5 µg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 µg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 µg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes.

Identification of Potentially Tolerated Fish Species by Multiplex IgE Testing of a Multinational Fish-Allergic Patient Cohort.

BACKGROUND: Although recent studies indicated that many fish-allergic patients may safely consume certain fish species, no clinical guidelines are available for identification of the exact species tolerated by specific patients. OBJECTIVE: To investigate whether multiplex immunoglobulin E (IgE) testing reveals potentially tolerated fish through absence of IgE to parvalbumin (PV) and extracts from specific species. METHODS: Sera from 263 clinically well-defined fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway, and Spain were used in a research version of the ALEX2 multiplex IgE quantification assay. Specific IgE to PVs from 10 fish species (9 bony and 1 cartilaginous), and to extracts from 7 species was quantified. The IgE signatures of individual patients and patient groups were analyzed using SPSS and R. RESULTS: Up to 38% of the patients were negative to cod PV, the most commonly used molecule in fish allergy diagnosis. Forty-five patients (17%) tested negative to PVs but positive to the respective fish extracts, underlining the requirement for extracts for accurate diagnosis. Between 60% (Spain) and 90% (Luxembourg) of the patients were negative to PV and extracts from ray, a cartilaginous fish, indicating its potential tolerance. Up to 21% of the patients were negative to at least 1 bony fish species. Of the species analyzed, negativity to mackerel emerged as the best predictive marker of negativity to additional bony fish, such as herring and swordfish. CONCLUSIONS: Parvalbumins and extracts from multiple fish species relevant for consumption should be used in fish-allergy diagnosis, which may help identify potentially tolerated species for individual patients.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection.

In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2Apro), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2Apro cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2Apro interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2Apro sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2Apro interaction is essential for successful viral replication. In contrast, HRV4 2Apro and coxsackievirus B4 2Apro failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.