Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes.

Novel approaches for the prevention of allergy are required, because of the inevitably increasing prevalence of allergic diseases during the last 30 years. Here, a recombinant chimeric protein, which comprises the whole amino acid sequences of three bee venom major allergens has been engineered and used in prevention of bee venom sensitization in mice. Phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 3) fragments with overlapping amino acids were assembled in a different order in the Api m (1/2/3) chimeric protein, which preserved entire T cell epitopes, whereas B cell epitopes of all three allergens were abrogated. Accordingly, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced in humans. Supporting these findings, the Api m (1/2/3) induced 100 to 1000 times less type-1 skin test reactivity in allergic patients. Treatment of mice with Api m (1/2/3) led to a significant reduction of specific IgE development towards native allergen, representing a protective vaccine effect in vivo. These results demonstrate a novel prototype of a preventive allergy vaccine, which preserves the entire T cell epitope repertoire, but bypasses induction of IgE against native allergen, and side effects related to mast cell/basophil IgE FcepsilonRI cross-linking in sensitized individuals.

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy.

BACKGROUND: Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. OBJECTIVE: The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. METHODS: By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). RESULTS: The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. CONCLUSION: The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.

Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides.

In allergy and asthma, the fine balance between the T helper (Th) 1, Th2 and T regulatory cytokine responses appears to be shifted towards Th2. Here, we report that synthetic lipopeptides which contain the typical lipid part of the lipoprotein of gram-negative bacteria stimulate a distinct regulatory cytokine pattern and inhibit several Th2 cell-related phenomena. The most potent analogue of synthetic lipopeptides, lipopeptide CGP 40774 (LP40) was not active in MyD88-deficient mice and stimulated Toll-like receptor (TLR)-2, but not TLR-4. LP40 potentiated the production of IFN-gamma and IL-10, but not IL-4 and IL-5 by human T cells. In addition, triggering of TLR-2 by lipopeptides promoted the in vitro differentiation of naive T cells towards IL-10- and IFN-gamma-producing T cells and suppressed IL-4 production by Th2 cells. Accordingly, LP40 inhibited IgE production induced by allergen, anti-IgD antibody, Nippostrongylus brasiliensis or murine acquired immunodeficiency virus. Furthermore, ovalbumin-induced lung eosinophilic inflammation was abolished and Schistosoma mansoni egg-induced granuloma size and eosinophil counts were suppressed in mice by LP40. These results demonstrate that stimulation of TLR-2 by lipopeptides represents a novel way for possible treatment of allergy and asthma by regulating the disrupted cytokine balance.