Allergen‐specific immunotherapy (AIT) is an allergen‐specific form of treatment for patients suffering from immunoglobulin E (IgE)‐associated allergy; the most common and important immunologically mediated hypersensitivity disease. AIT is based on the administration of the disease‐causing allergen with the goal to induce a protective immunity consisting of allergen‐specific blocking IgG antibodies and alterations of the cellular immune response so that the patient can tolerate allergen contact. Major advantages of AIT over all other existing treatments for allergy are that AIT induces a long‐lasting protection and prevents the progression of disease to severe manifestations. AIT is cost effective because it uses the patient´s own immune system for protection and potentially can be used as a preventive treatment. However, broad application of AIT is limited by mainly technical issues such as the quality of allergen preparations and the risk of inducing side effects which results in extremely cumbersome treatment schedules reducing patient´s compliance. In this article we review progress in AIT made from its beginning and provide an overview of the state of the art, the needs for further development, and possible technical solutions available through molecular allergology. Finally, we consider visions for AIT development towards prophylactic application.

Background: Macrophages can be converted in vitro into immunoregulatory M2b macrophages in the presence of immune complexes ( IC s), but the role of the specific subclasses IgG1 or IgG4 in this phenotypic and functional change is not known. Objective: We aimed to refine the original method by applying precisely defined IC s of the subclasses IgG4 or IgG1 constructed by two independent methods. Methods: Monocyte‐derived macrophages ( MDM s) were treated with M‐ CSF , followed by IL ‐4/ IL ‐13 to induce the M2a allergic phenotype. To mimic unspecific or allergen‐specific IC s, plates were coated with myeloma IgG1 or IgG4, or with grass pollen allergen Phl p 5 followed by recombinant human Phl p 5‐specific IgG1 or IgG4. M2a polarized macrophages were then added, cultured, and examined for cellular markers and cytokines by flow cytometry, ELISA , and rt PCR . Alternatively, immune complexes with IgG1 or IgG4 were formed using protein L. Results: IgG4 IC s down regulated CD 163 and CD 206 on M2a cells, and significantly increased IL ‐10, IL ‐6, TNF α, and CCL 1 secretion, indicating a shift to an M2b‐like phenotype. Treatment with IgG4 IC s resulted in expression of Fcγ RII and down modulation of Fcγ RII compared with IgG1 treated cells ( P = 0.0335) or untreated cells ( P < 0.00001). Conclusion: Immune complexes with subclasses IgG1 and IgG4 can in vitro be generated by plate absorption, and in fluid form by protein L. Cross‐linking of Fcγ RII b by the IgG4 subclass redirects pro‐allergic M2a macrophages to an M2b‐like immunosuppressive phenotype. This suggests an interplay of macrophages with IgG4 in immune tolerance, likely relevant in allergen immunotherapy.

IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans.

The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10^-10 M) and had slow off-rates (10^-4 s^-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

Background: Recent studies showed that a single injection of human monoclonal allergen‐specific IgG antibodies significantly reduced allergic symptoms in birch pollen‐allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen‐specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE‐mediated responses.Methods: A cDNA‐VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope‐specificity and affinity to Bet v 1. Furthermore, cross‐reactivity to Bet v 1‐homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen‐induced basophil activation were investigated.Results: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross‐react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha‐helix at the C‐terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1‐induced basophil activation.Conclusion: We identified high‐affinity Bet v 1‐specific nanobodies that recognize an important IgE epitope and reduce allergen‐induced basophil activation revealing the first proof that allergen‐specific nanobodies are useful tools for future treatment of pollen allergy.