Molecular Evolution of Hypoallergenic Hybrid Proteins for Vaccination against Grass Pollen Allergy

Abstract

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A–adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients’ IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

Newly designed hemagglutinin-Der p 2 chimera is a potential candidate for allergen specific immunotherapy

“…However, in spite of beneficial clinical efficacy, recombinant allergen preparations revealed poor immunogenicity, and therefore they do not induce significantly better therapeutic effects than natural extracts [8,9]. Moreover, there is a need for employment of novel immunomodulators capable to enhance the innate immune response in order to economically justify the production of recombinant allergy vaccines [10].…”

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant

“…7). In all of the four sera, it was observed that TYKW-mut did not reach an IC 50 value at a concentration as high as 10 4 ng in 100 l of each serum (i.e. 100 g/ml).…”

“…H&E and PAS staining of lung histology were done to quantify the eosinophilic infiltrates and mucous gland hyperplasia, respectively, as described (49). For raising antisera, three BALB/c mice (10 g of antigens/mouse) and one New Zealand White rabbit (200 g of antigens/rabbit) were immunized with either rRhi o 1 or TYKW-mut adsorbed to Freund's adjuvants following protocols described previously (50). About 3-4 mg of IgG were purified from rabbit sera (preimmune and immune) using a Pierce TM Protein A IgG purification kit.…”

Modulation of the specific immune response in Balb/c mice by intranasal application of recombinant H1D2 chimera

“…In another study, a clinical improvement was observed by treatment with the recombinant birch pollen allergen Bet v 1 . Although clinical trials with recombinant allergen preparations revealed good clinical efficacy, their poor immunogenicity represents a problem, as it does not induce significantly better therapeutic effects than natural extracts . Therefore, the application of novel immunomodulators capable of activating the innate immune response might economically justify the production of recombinant allergy vaccines …”