Recombinant PreS-fusion protein vaccine for birch pollen and apple allergy.

BACKGROUND: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. METHODS: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA. RESULTS: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV. CONCLUSION: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

BACKGROUND: The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. OBJECTIVE: To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. METHODS: Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. RESULTS: IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. CONCLUSION: IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Novel vaccines for allergen-specific immunotherapy.

PURPOSE OF REVIEW: Allergen-specific immunotherapy (AIT) is a highly economic, effective and disease-modifying form of allergy treatment but requires accurate prescription and monitoring. New molecular approaches are currently under development to improve AIT by reducing treatment-related side effects, cumbersome protocols and patients' compliance. We review the current advances regarding refined diagnosis for prescription and monitoring of AIT and the development of novel molecular vaccines for AIT. Finally, we discuss prophylactic application of AIT. RECENT FINDINGS: There is evidence that molecular allergy diagnosis not only assists in the prescription and monitoring of AIT but also allows a refined selection of patients to increase the likelihood of treatment success. New data regarding the effects of AIT treatment with traditional allergen extracts by alternative routes have become available. Experimental approaches for AIT, such as virus-like particles and cell-based treatments have been described. New results from clinical trials performed with recombinant hypoallergens and passive immunization with allergen-specific antibodies highlight the importance of allergen-specific IgG antibodies for the effect of AIT and indicate opportunities for preventive allergen-specific vaccination. SUMMARY: Molecular allergy diagnosis is useful for the prescription and monitoring of AIT and may improve the success of AIT. Results with molecular allergy vaccines and by passive immunization with allergen-specific IgG antibodies indicate the importance of allergen-specific IgG capable of blocking allergen recognition by IgE and IgE-mediated allergic inflammation as important mechanism for the success of AIT. New molecular vaccines may pave the road towards prophylactic allergen-specific vaccination.

Preventive Administration of Non-Allergenic Bet v 1 Peptides Reduces Allergic Sensitization to Major Birch Pollen Allergen, Bet v 1.

IgE-mediated allergy to birch pollen affects more than 100 million patients world-wide. Bet v 1, a 17 kDa protein is the major allergen in birch pollen responsible for allergic rhinoconjunctivitis and asthma in birch pollen allergic patients. Allergen-specific immunotherapy (AIT) based on therapeutic administration of Bet v 1-containing vaccines is an effective treatment for birch pollen allergy but no allergen-specific forms of prevention are available. We developed a mouse model for IgE sensitization to Bet v 1 based on subcutaneous injection of aluminum-hydroxide adsorbed recombinant Bet v 1 and performed a detailed characterization of the specificities of the IgE, IgG and CD4+ T cell responses in sensitized mice using seven synthetic peptides of 31-42 amino acids length which comprised the Bet v 1 sequence and the epitopes recognized by human CD4+ T cells. We then demonstrate that preventive systemic administration of a mix of synthetic non-allergenic Bet v 1 peptides to 3-4 week old mice significantly reduced allergic immune responses, including IgE, IgG, IgE-mediated basophil activation, CD4+ T cell and IL-4 responses to the complete Bet v 1 allergen but not to the unrelated major grass pollen allergen Phl p 5, without inducing Bet v 1-specific allergic sensitization or adaptive immunity. Our results thus demonstrate that early preventive administration of non-allergenic synthetic T cell epitope-containing allergen peptides could be a safe strategy for the prevention of allergen-specific IgE sensitization.

Microarray-Based Allergy Diagnosis: Quo Vadis?

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.