Milena Weber

Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy

Background We have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier. Methods We conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n = 70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4 weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment. Results Sixty-eight patients completed the trial. TNSS significantly decreased with mean changes of − 1.41 (BM32/20 μg) (P = 0.03) and − 1.34 (BM32/40 μg) (P = 0.003) whereas mean changes in the BM32/10 μg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P < 0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P = 0.0063). Conclusion The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.)

Conversion of Der p 23, a New Major House Dust Mite Allergen, into a Hypoallergenic Vaccine

Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus–derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23–specific IgG Abs that inhibited binding of patients’ IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23–induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-γ in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.

Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction

The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction.

The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes

Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes.

IgE epitope proximity determines immune complex shape and effector cell activation capacity

Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases.

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

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.

Safety and efficacy of immunotherapy with the recombinant B-cell epitope–based grass pollen vaccine BM32

Background: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE‐binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. Objective: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen–induced rhinitis and controlled asthma. Methods: A double‐blind, placebo‐controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 &mgr;g, n = 60) or high dose (160 &mgr;g, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo‐treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen‐specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. Results: Although statistical significance regarding the primary end point was not reached, BM32‐treated subjects, when compared with placebo‐treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen‐specific IgG without induction of allergen‐specific IgE and a reduction in the seasonally induced increase in allergen‐specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. Conclusions: Injections of BM32 induced allergen‐specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

Comparison of the immunogenicity of BM32, a recombinant hypoallergenic B cell epitope–based grass pollen allergy vaccine with allergen extract–based vaccines

Allergen-specific immunotherapy (AIT) is a clinically and cost-effective allergy treatment that modifies the course of the disease and has long-lasting effects.1,E7,E8 However, allergen extract–based forms of AIT require administration of multiple doses, which makes treatment cumbersome and leads to poor compliance in patients.2 A number of approaches were proposed to address this issue, including use of AIT materials with higher safety, such as allergoids, recombinant allergen derivatives, and allergen-derived peptides, which allow shortening of the buildup phase.E9

Extracorporeal IgE Immunoadsorption in Allergic Asthma: Safety and Efficacy

Background Prevention of IgE-binding to cellular IgE-receptors by anti-IgE (Omalizumab) is clinically effective in allergic asthma, but limited by IgE threshold-levels. To overcome this limitation, we developed a single-use IgE immunoadsorber column (IgEnio). IgEnio is based on a recombinant, IgE-specific antibody fragment and can be used for the specific extracorporeal desorption of IgE. Objective To study safety and efficacy of IgEnio regarding the selective depletion of IgE in a randomized, open-label, controlled pilot trial in patients with allergic asthma and to investigate if IgEnio can bind IgE-Omalizumab immune complexes. Methods Fifteen subjects were enrolled and randomly assigned to the treatment group (n = 10) or to the control group (n = 5). Immunoadsorption was done by veno-venous approach, processing the twofold calculated plasma volume during each treatment. A minimum average IgE-depletion of 50% after the last cycle in the intention-to-treat population was defined as primary endpoint. Safety of the treatment was studied as secondary endpoint. In addition, possible changes in allergen-specific sensitivity were investigated, as well as clinical effects by peak flow measurement and symptom-recording. The depletion of IgE-Omalizumab immune complexes was studied in vitro. The study was registered at clinicaltrials.gov (NCT02096237) and conducted from December 2013 to July 2014. Results IgE immunoadsorption with IgEnio selectively depleted 86.2% (± 5.1% SD) of IgE until the end of the last cycle (p < 0.0001). Removal of pollen allergen-specific IgE was associated with a reduction of allergen-specific basophil-sensitivity and prevented increases of allergen-specific skin-sensitivity and clinical symptoms during pollen seasons. IgEnio also depleted IgE-Omalizumab immune complexes in vitro. The therapy under investigation was safe and well-tolerated. During a total of 81 aphereses, 2 severe adverse events (SAE) were recorded, one of which, an episode of acute dyspnea, possibly was related to the treatment and resolved after administration of antihistamines and corticosteroids. Conclusions This pilot study indicates that IgE immunoadsorption with IgEnio may be used to treat patients with pollen-induced allergic asthma. Furthermore, the treatment could render allergic patients with highly elevated IgE-levels eligible for the administration of Omalizumab and facilitate the desorption of IgE-Omalizumab complexes. This study was funded by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.

Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet

Background Celiac disease (CD) is an inflammatory disease of the small intestine caused by an immunologic hypersensitivity reaction to dietary wheat gluten. Objectives We sought to clone, express, and perform IgA epitope mapping of a CD-specific wheat antigen and to study its usefulness for identifying patients with CD and monitoring adherence to a gluten-free diet. Methods A synthetic gene coding for γ-gliadin 1 (GG1) was expressed in Escherichia coli. Recombinant γ-gliadin 1 (rGG1) was purified and characterized biochemically, structurally, and immunologically by using sera from patients with CD and control subjects. Overlapping GG1 peptides were synthesized for IgA and IgG epitope mapping. GG1 and peptide-specific antibodies were raised for tracing GG1 in cereals and dietary wheat products and to study its resistance to digestion. Results rGG1 was expressed and purified. rGG1-based IgA ELISAs performed in populations of patients with CD and control subjects showed a specificity of 92.9%, which was higher than that of gliadin extract (e). Furthermore, it allowed monitoring of adherence to a gluten-free diet in patients. A 26-amino-acid peptide from the proline-glutamine–rich repetitive N-terminal region was identified as the immunodominant IgA epitope. GG1-related antigens were found in rye, barley, and spelt but not in oat, rice, or maize. GG1 was detected in dietary wheat products after baking, and in particular, the major IgA epitope–containing region was resistant against digestion. Conclusions rGG1 and its epitope might be useful for identifying patients with CD, monitoring treatment, and studying the pathomechanisms of CD and development of preventive and therapeutic strategies.