Two years of treatment with the recombinant grass pollen allergy vaccine BM32 induces a continuously increasing allergen-specific IgG4 response.

BACKGROUND: BM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32. METHODS: Blood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs). FINDINGS: Two years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients. INTERPRETATION: AIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses. FUNDINGS: Grants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF).

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 µg, n = 60) or high dose (160 µg, 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.

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 4weeks 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 (BM32/20µg) (P=0.03) and -1.34 (BM32/40µg) (P=0.003) whereas mean changes in the BM32/10µg 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.).

Frequent occurrence of T cell-mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments.

BACKGROUND: Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity. OBJECTIVE: We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD). METHODS: A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)(+) and CCR4(+) T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively. RESULTS: rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1-specific proliferation of CLA(+) and CCR4(+) T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1-specific CLA(+) and CCR4(+) proliferation and cytokine secretion in patients with and without APT reactions. CONCLUSION: Hypoallergenic rBet v 1 fragments induce T cell-dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.

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.

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy.

BACKGROUND: Grass pollen is one of the most important sources of respiratory allergies worldwide. OBJECTIVE: This study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach. METHODS: Fusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity. RESULTS: Ten hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation. CONCLUSION: A recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly.

BACKGROUND: At least 100 million patients suffer from birch pollen allergy. OBJECTIVE: Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. METHODS: Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. RESULTS: Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. CONCLUSION: We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch pollen allergy.

The influence of recombinant production on the immunologic behavior of birch pollen isoallergens.

BACKGROUND: Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity. METHODOLOGY: Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity. CONCLUSION: The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.