Frank Stolz

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

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.)

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

Background Fish is a frequent elicitor of severe IgE‐mediated allergic reactions. Beside avoidance, there is currently no allergen‐specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium‐binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. Methods C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T‐cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti‐hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1–specific basophil degranulation, and Cyp c 1–induced allergic symptoms in the mouse model. Results A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1–induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Conclusions Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish 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.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

Antibodies induced by immunization with a hypoallergenic mutant of the major fish allergen Cyp c 1 inhibit allergic symptoms in a mouse model of fish allergy

Background Fish allergy is one of the most frequently occurring allergies to animal-derived food and may cause severe anaphylactic reactions. Currently allergen-specific immunotherapy for fish allergy is not available as it may induce severe side-effects. A hypoallergenic mutant of the major carp allergen Cyp c 1 (mCyp c 1) has recently been developed for the treatment of IgE-mediated fish allergy. We analysed the effect of antibodies induced by immunization with mCyp c 1 on allergic symptoms in a mouse model of fish allergy.